TW202222832A - Anti-integrin beta7 antibody formulations and devices - Google Patents

Abstract

Formulations comprising an anti-integrin beta7 antibody or an antigen-binding fragment thereof are provided, including pharmaceutical formulations. Also provided are article of manufactures comprising such formulations, and methods of using such formulations.

Description

Anti-integrin beta7 antibody formulations and devices

Formulations, including pharmaceutical formulations, comprising anti-integrin beta7 antibodies or antigen-binding fragments thereof, and devices comprising the formulations and methods of using the formulations and devices are provided.

Integrins are alpha beta heterodimeric cell surface receptors involved in numerous cellular processes from cell adhesion to gene regulation (Hynes Cell (1992); 69: 11-25; and Hemler, Annu. Rev. Immunol . (1990) ), 8:365-368). Several integrins have been implicated in disease processes and have attracted considerable attention as potential targets for drug development (Sharar et al., Springer Semin. Immunopathol . (1995); 16:359-378). In the immune system, integrins are involved in leukocyte transport, adhesion and infiltration during inflammatory processes (Nakajima et al., J. Exp. Med . (1994); 179:1145-1154). Different manifestations of integrins can regulate the adhesive properties of cells and different integrins are involved in different inflammatory responses (Butcher et al., Science (1996); 272:60-66. β7 integrins (ie α4β7 and αEβ7) are mainly manifested On monocytes, lymphocytes, eosinophils, basophils, and macrophages, but not on neutrophils (Elices et al., Cell (1990); 60:577-584). α4β7 integral Major coordination systems for catenin endothelial surface proteins mucosal addressin cell adhesion molecule (MAdCAM) and vascular cell adhesion molecule (VCAM-1) (Makarem et al., J. Biol. Chem . (1994);269:4005-4011) Binding of α4β7 to MAdCAM and/or VCAM expressed on high endothelial venules (HEVs) at the site of inflammation results in firm leukocyte adhesion to the endothelium and subsequent extravasation into inflamed tissue (Chuluyan et al., Springer Semin. Immunopathol . , 1995, 16:391404). Monoclonal antibodies against α4β7, MAdCAM or VCAM have been shown to be potent modulators in animal models of chronic inflammatory diseases such as asthma (Laberge et al., Am. J. Respir. (1995); 151: 822-829 .), rheumatoid arthritis (Barbadillo et al., Springer Semin. Immunopathol. (1995);16:375-379), colitis (Viney, J. (1996); 157: 2488-2497) and inflammatory bowel disease (Podalski, N. Eng. J. Med . (1991); 325: 928-937; Powrie et al., Ther. Immunol . (1995); 2:115-123).

Humanized anti-integrin beta7 antibodies and antigen-binding fragments thereof have been described. See, eg , International Patent Publication No. WO2006/026759. A specific anti-integrin beta7 antibody, etrolizumab, has been studied clinically for the treatment of inflammatory disorders of the gastrointestinal tract, such as inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. disease). The results of a phase 1 study of ivolizumab in moderate to severe ulcerative colitis are described in Rutgeerts PJ et al. Gut 2013;62:1122–1130, which reports that no clinically significant safety signal has been observed. Results of a global, multicenter Phase 2 study of evolizumab in moderate-to-severe ulcerative colitis demonstrate evidence of clinical efficacy of evolizumab treatment, as measured by induction of clinical remission (Vermeire, S. . et al, Lancet 2014; 384: 309–18). In addition, clinically meaningful responses were observed following the use of etrolizumab in patients with moderate to severe Crohn's disease (Sandborn et al., presentation titled "Etrolizumab as Induction Therapy in Moderate to Severe Crohn's Disease: Results from Bergamot Cohort 1", presented at United European Gastroenterology Week Congress, Oct. 28-Nov. 2, 2017). Accordingly, evolizumab has demonstrated promise as a therapeutic treatment option in inflammatory bowel disease and other studies are improving the safety and efficacy profile of evolizumab.

In every reported study to date, ivolizumab was administered intravenously or subcutaneously by a healthcare provider in a clinical setting. For subcutaneous administration, vials and syringes were used and the vial concentration was 150 mg/ml. Because inflammatory bowel diseases (eg, ulcerative colitis and Crohn's disease) are chronic diseases, long-term therapeutic treatment with ivolizumab may be required. For optimal patient convenience and compliance, among other advantages, it is desirable to self-administer irolizumab or to administer it at home by a caregiver or healthcare professional. Accordingly, it would be advantageous to develop self-administered devices and formulations of ilrolizumab that are compatible with such devices.

Because proteins (including antibodies, such as ilozumab) are larger and more complex (eg, possess multiple functional groups in addition to complex three-dimensional structures) than traditional organic and inorganic drugs, the formulation of these proteins can cause special problems. question. In order for a protein to remain biologically active, the formulation must maintain intact at least the conformational integrity of the core sequence of the protein's amino acids, while simultaneously protecting multiple functional groups of the protein from degradation. Degradation pathways for proteins can involve chemical instability (eg, any process involving modification of a protein by bond formation or cleavage to generate new chemical entities) or physical instability (eg, changes in the higher order structure of a protein). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta elimination, or disulfide exchange. Physical instability can arise from, for example, denaturation, aggregation, precipitation, or adsorption. The three most common protein degradation pathways are protein aggregation, deamidation, and oxidation. Cleland et al, Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993).

Liquid antibody formulations at high concentrations (eg > 100 mg/mL) may be desirable, eg, for therapeutic routes of administration or for therapeutic applications, where small volumes of drug product, eg, for subcutaneous injection, may be appropriate, including, eg, using Prefilled syringes or self-administering devices. However, high concentration antibody formulations can pose numerous challenges and problems, including those associated with the use of pre-filled syringes or self-administration devices. One problem is the instability caused by the formation of particles. In the case of reconstituted liquid formulations, this problem has been addressed by the use of surfactants such as polysorbates, but surfactants are sometimes considered unsuitable for liquid formulations because This makes further processing more difficult. In addition, the surfactant additionally does not reduce the viscosity increase due to the many intermolecular interactions resulting from the macromolecular properties of the antibody.

Selection of pH and optimal excipients is important for preventing particle formation due to polysorbate-induced degradation, preventing isomerization of certain amino acids and formation of undesired intermediates, and prolonged storage longevity, as well as providing manufacturing advantages. Selection of pH and optimal excipients is also important for formulation development, for example, for compatibility with storage conditions; administration by prefilled syringes, including devices containing prefilled syringes (e.g., with needle safety); pre-filled syringes of the device) or auto-injectors or self-administration devices, for example to ensure compatibility with device components and to provide low injection forces.

It is highly advantageous that formulations comprising anti-beta7 antibodies, including ivolizumab, have prolonged stability and low viscosity at high antibody concentrations. High antibody concentration formulations with these properties are highly advantageous for certain routes of administration (eg, subcutaneous administration), including use with prefilled syringes and self-administration devices. The formulations provided herein address these needs and provide other useful benefits.

It is highly advantageous that formulations comprising anti-beta7 antibodies have prolonged stability and low viscosity at high antibody concentrations. High antibody concentration formulations with these properties are highly advantageous for certain routes of administration, such as subcutaneous administration. The formulations provided herein address these needs and provide other useful benefits.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose.

The formulations of the present disclosure are based, at least in part, on the discovery that the anti-integrin β7 antibody irolizumab described herein can be formulated at high concentrations (approximately > 100 mg/mL) in histidine buffer and arginine succinates and surfactants, and the high antibody concentration formulations have low viscosity, have prolonged physical and chemical stability, and maintain efficacy. The formulations disclosed herein are best compatible with self-administration devices, such as prefilled syringes with needle safety devices (PFS-NSDs). In certain embodiments, prefilled syringes are assembled into autoinjectors. The formulations disclosed herein can be packaged into subcutaneous administration devices that maintain product stability and other desirable attributes as described herein. Formulations of the present disclosure can be used, for example, to treat inflammatory disorders of the gastrointestinal tract, such as inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease.

Accordingly, in one aspect, formulations comprising an anti-integrin beta7 antibody or antigen-binding fragment thereof are provided. In certain embodiments, the concentration of the antibody or antigen-binding fragment thereof in the formulation is at least about 100 mg/mL and the viscosity of the formulation at 25°C is less than about 20 centipoise (cP). In certain embodiments, the formulation has a viscosity of less than about 7 cP at 25°C.

In certain embodiments, the anti-integrin beta7 antibody is a monoclonal antibody. In certain embodiments, the anti-integrin beta7 antibody is a humanized antibody. In certain embodiments, an anti-integrin beta7 antibody or antigen-binding fragment thereof comprises three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, where: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain variable region, which Contains the amino acid sequence shown in SEQ ID NO:9. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 11 The amino acid sequence shown in . In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 12 The amino acid sequence shown in . In certain embodiments, the anti-integrin beta7 antibody is ilozumab. In certain embodiments, the concentration of the anti-integrin beta7 antibody or antigen-binding fragment thereof in the formulation is between about 100 mg/ml and about 220 mg/ml. In certain embodiments, the concentration of anti-integrin beta7 antibody or antigen-binding fragment thereof in the formulation is about 150 mg/ml.

In certain embodiments, the pH of the formulation is greater than 5.0 and up to 7.0. In certain embodiments, the pH of the formulation is greater than 5.5. In certain embodiments, the pH of the formulation is between 5.6 and 6.1. In certain embodiments, the pH of the formulation is 5.8, between 5.7 and 5.9, or between 5.75 and 5.85.

In certain embodiments, the formulation comprises arginine-succinate. In certain embodiments, the concentration of arginine-succinate in the formulation is between about 100 mM and about 300 mM. In certain embodiments, the concentration of arginine-succinate in the formulation is between about 150 mM and about 300 mM. In certain embodiments, the concentration of arginine-succinate in the formulation is between about 150 mM and about 250 mM. In certain embodiments, the concentration of arginine succinate in the formulation is about 200 mM.

In certain embodiments, the formulation further comprises a surfactant, and the concentration of the surfactant in the formulation is greater than 0.01% weight/volume (w/v) and up to about 1% w/v. In certain embodiments, the concentration of surfactant in the formulation is between 0.03% w/v and 0.06% w/v. In certain embodiments, the concentration of surfactant in the formulation is at or about 0.04% w/v. In certain embodiments, the surfactant is polysorbate 20.

In certain embodiments, the formulation further comprises histidine. In certain embodiments, the concentration of histidine in the formulation is between about 5 mM and about 40 mM. In certain embodiments, the concentration of histidine in the formulation is at or about 20 mM.

In certain embodiments, the formulations have prolonged stability. In certain embodiments, the anti-integrin beta7 antibody is stable at -20°C for at least about 7 years. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is stable at 5°C for at least about one year. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is stable at 5°C for at least about 5 years. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is stable for about 6 years at 5°C.

In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is stable at room temperature for at least about 1 day. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is stable at room temperature for up to about one month.

In another aspect, the present disclosure provides a formulation comprising an anti-integrin beta7 antibody or antigen-binding fragment thereof, 20 mM or about 20 mM histidine buffer (pH 5.8), 0.04% polysorbate 20, and 200 mM or about 200 mM arginine succinate, wherein the anti-integrin β7 antibody is at a concentration of about 150 mg/ml, and wherein the anti-integrin β7 antibody comprises three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, of which: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

In certain embodiments, the formulation has a pH between 5.7 and 5.9 or between 5.75 and 5.85. In certain embodiments, the formulation comprises 0.04% polysorbate 20 at or about 0.04% polysorbate 20.

The subject matter disclosed herein provides a formulation comprising an anti-integrin beta7 antibody in 20 mM histidine buffer at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 or about 20 mM histidine buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein anti- The integrin beta7 antibody comprises three light chain hypervariable regions (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein : (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises The amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence SEQ ID NO: 5; and (vi) HVR-H3 comprises the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In certain embodiments, the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising the amine set forth in SEQ ID NO: 11 base acid sequence. In certain embodiments, the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO:10; and a heavy chain comprising the amine set forth in SEQ ID NO:12 base acid sequence. In certain embodiments, the anti-integrin beta7 antibody is ilozumab.

In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain variable region, which Contains the amino acid sequence shown in SEQ ID NO:9. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 11 The amino acid sequence shown in . In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 12 The amino acid sequence shown in .

In yet another aspect, an article of manufacture comprising a subcutaneous administration device is provided. In certain embodiments, a subcutaneous delivery device delivers a fixed dose of an anti-integrin beta7 antibody or antigen-binding fragment thereof to a subject. In certain embodiments, the fixed dose is about 100 mg. In certain embodiments, the fixed dose is 105 mg. In certain embodiments, the fixed dose is about 200 mg. In certain embodiments, the fixed dose is 210 mg. In certain embodiments, the anti-integrin beta7 antibody is ilozumab. The anti-integrin beta7 antibody or antigen-binding fragment thereof is formulated in a subcutaneous administration device as described above, thereby providing it in a stable pharmaceutical formulation.

In certain embodiments, the subcutaneous administration device is a needle safety device. In certain embodiments, the needle safety device comprises a prefilled syringe. In certain embodiments, the anti-integrin beta7 antibody is stable at 5°C for at least about 60 months at a subcutaneous administration device, or at 25°C for at least about 3 months. In certain embodiments, a prefilled syringe includes a glass barrel, a plunger, a needle, and a needle shield or cap. In certain embodiments, the needle shield is a rigid needle shield. In certain embodiments, the rigid needle shield comprises a rubber formulation with low zinc content. In certain embodiments, the rigid needle shield includes a resilient member and a rigid shield. In certain embodiments, prefilled syringes are assembled into autoinjectors.

In certain embodiments, the volume of the formulation contained in the prefilled syringe is between about 0.5 mL and about 2.0 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is between about 0.5 mL and about 1.0 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 0.7 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 0.75 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.0 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is between about 1.0 mL and about 1.5 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.4 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.5 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.45 mL. In certain embodiments, the prefilled syringe has a syringe capacity of 1 mL. In certain embodiments, the prefilled syringe has a syringe capacity of 2.25 mL.

In certain embodiments, the prefilled syringe contains silicone oil. In certain embodiments, the amount of silicone oil in the prefilled syringe is no greater than about 1 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is between about 0.1 mg and about 1 mg. In certain embodiments, wherein the amount of silicone oil in the prefilled syringe is between about 0.2 mg and about 0.6 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is between about 0.5 mg and 0.9 mg.

In certain embodiments, the needle safety device has an injection force of no greater than about 50 Newtons (N). In certain embodiments, the needle safety device has an injection force of no greater than about 35 Newtons (N). In certain embodiments, the needle safety device has an injection force of no greater than about 33 Newtons (N).

The subject matter disclosed herein provides an article of manufacture comprising about 0.7 mL of the formulation and a subcutaneous administration device, wherein (a) a formulation comprising an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody comprises three One light chain hypervariable region (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) A subcutaneous administration device is a needle safety device comprising a 1 mL prefilled syringe with a syringe capacity of 1 mL.

In certain embodiments, the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL.

The subject matter disclosed herein provides an article of manufacture comprising about 1.4 mL of the formulation and a subcutaneous administration device, wherein (a) Formulation comprising anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody comprises three One light chain hypervariable region (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The hypodermic delivery device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 2.25 mL.

In certain embodiments, the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL.

The subject matter disclosed herein further provides auto-injectors comprising the articles disclosed herein.

The subject matter disclosed herein provides an auto-injector comprising an article of manufacture comprising about 0.7 mL of the formulation and a subcutaneous administration device, wherein (a) Formulation comprising anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody comprises three One light chain hypervariable region (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) A subcutaneous administration device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 1 mL.

In certain embodiments, the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL.

The subject matter disclosed herein provides an auto-injector comprising an article of manufacture containing about 1.4 mL of the formulation and a subcutaneous administration device, wherein (a) Formulation comprising anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody comprises three One light chain hypervariable region (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) HVR-H2 comprises the amino acid sequence of SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The hypodermic delivery device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 2.25 mL.

In certain embodiments, the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL.

In yet another aspect, a method of treating an inflammatory disorder of the gastrointestinal tract in a subject is provided. In certain embodiments, the method comprises administering to the subject an effective amount of any of the formulations described above.

In yet another aspect, a method of subcutaneously administering a formulation comprising an anti-integrin beta7 antibody or antigen-binding fragment thereof is provided. Such methods comprise subcutaneous administration of any of the formulations described above. In certain embodiments, the methods comprise a subcutaneous administration device according to any of the devices described above. In certain embodiments, the method comprises the auto-injector disclosed herein. In certain embodiments, the administration results in reduced or no pain. In certain embodiments, administration produces transient and mild injection site reactions. In certain embodiments, the full dose is administered or at least 90% of the full dose is administered. In certain embodiments, administration provides an exposure to ilrolizumab equivalent to a pre-filled syringe with a needle safety device.

The subject matter disclosed herein also provides use of the formulations, articles of manufacture, or auto-injectors disclosed herein, in therapy.

In addition, the subject matter disclosed herein provides the use of the formulations, articles of manufacture, or auto-injectors disclosed herein for treating an inflammatory disorder of the gastrointestinal tract in a subject. In certain embodiments, the gastrointestinal inflammatory disorder is inflammatory bowel disease. In certain embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

Cross-references to related applications

This application claims priority from U.S. Provisional Patent Application No. 63/059,427, filed on July 31, 2020, the contents of which are incorporated by reference in their entirety and from which priority is claimed. sequence listing

This application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. This ASCII copy was created on July 27, 2021, named 00B2061132SL.txt and is 13,697 bytes in size.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th edition, John Wiley & Sons (New York , N.Y. 1992) provides general guidance on many of the terms used in this application to those skilled in the art.

For the purpose of interpreting this specification, the following definitions will apply and where appropriate, terms used in the singular will also include the plural and vice versa. To the extent that any definitions set forth below conflict with any document incorporated herein by reference, the definitions set forth below shall control.

As used in this specification and the appended claims, the singular forms "a (a/an)" and "the (the)" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" or "antibody" includes a plurality of proteins or antibodies, respectively; reference to a "cell" includes a mixture of cells, and the like.

As used herein, the term "about" or "approximately" means that a particular value is within an acceptable error range as determined by one of ordinary skill in the art to which this invention pertains, depending in part on how the value is measured or determined, i.e. , depending on the limitations of the measurement system . For example, "about" means 3 times or more the standard deviation, as is practiced in the art to which this invention pertains. Alternatively, "about" means a range of at most 20%, more preferably at most 10%, more preferably at most 5%, and more preferably at most 1% of the given value . Alternatively, particularly with respect to biological systems or processes, the term means within an order of magnitude of the numerical value, optimally within 5 times the numerical value, and more preferably within 2 times the numerical value. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that is free of other components that would be unacceptably toxic to the individual to which the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to an individual mammal to provide an effective dose of the active ingredient employed.

A "sterile" formulation is one that is sterile or free or substantially free of all living microorganisms and their spores.

"Frozen" formulations are those with a temperature below 0°C. Typically, frozen formulations are not lyophilized, and they have not undergone prior or subsequent lyophilization. In certain embodiments, the refrigerated formulation comprises a refrigerated drug substance for storage (in a stainless steel tank) or a refrigerated drug product (in a final vial configuration).

A "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity upon storage. In certain embodiments, the formulations substantially retain their physical and chemical stability and their biological activity upon storage. The shelf life is generally selected based on the expected shelf life of the formulation.

As used herein, a formulation with "prolonged stability" means one in which the protein substantially retains its physical stability, chemical stability and biological activity when stored at 5°C for one year or more. In certain embodiments, the storage system is maintained at 5°C for one year or more. In certain embodiments, the storage system is maintained at 5°C for up to 5 or 6 years. In certain embodiments, the anti-integrin beta7 antibody is stable at room temperature for at least about 1 day. In certain embodiments, anti-integrin beta7 antibodies are stable at room temperature for up to about one month. As used herein, room temperature is between about 20°C and about 22°C. In certain embodiments, the room temperature is about 20°C.

If the protein in the pharmaceutical formulation exhibits no or minimal signs of aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or when measured by UV light scattering or by particle size sieve chromatography, then it "retains its physical stability".

A protein in a pharmaceutical formulation "retains its chemical stability" if its chemical stability over a given period of time is such that it can be considered to retain its biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of proteins. Chemical alterations may involve size adjustment (eg, trimming), which may be performed using, eg, particle size sieve chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) Evaluate. Other types of chemical changes include charge changes (eg, due to deamidation), which can be assessed, for example, by ion exchange chromatography or imaging capillary isoelectric focusing (icIEF).

An antibody in a pharmaceutical formulation "retains its biological activity" if its biological activity at a given time differs by about 20% (within assay error) from the biological activity at which the pharmaceutical formulation was prepared, eg, as in antigen binding test or efficacy test.

As used herein, the "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind an antigen. It may further include that the antibody binds to the antigen and produces a measurable biological response that is measurable in vitro or in vivo. Such activities can be antagonistic or agonistic.

The formulated antibody is substantially pure and desirably substantially homogeneous (eg, free of contaminating proteins, etc.). "Substantially pure" antibody means a composition comprising at least about 90% or at least about 95% by weight antibody, based on the total weight of the composition. An antibody that is "substantially homogeneous" means a composition comprising at least about 99% by weight of the antibody, based on the total weight of the composition.

"Isotonic" means that the formulation of interest has substantially the same osmolarity as human blood. Isotonic formulations typically have an osmolarity of about 250 mOsm to 350 mOsm. Isotonicity can be measured using, for example, a vapor pressure or frozen-type osmometer.

As used herein, "buffer" refers to a buffered solution that resists changes in pH through the action of an acid-base conjugate component.

As used herein, "surfactant" refers to a surfactant, typically a nonionic surfactant. Examples of surfactants herein include polysorbates (eg, polysorbate 20 and polysorbate 80); poloxamers (eg, poloxamer 188); Triton; lauryl sulfate Sodium (SDS); Sodium Lauryl Sulfate; Sodium Octyl Glycosides; Lauryl-, Myristyl-, Linoleyl- or Stearyl Sulfobetaine; Lauryl-, Myristyl-, Linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetylbetaine; lauroamidopropyl-, cocoamidopropyl-, linoleamidopropyl- , myristamidopropyl-, palmamidopropyl-, or isostearylamidopropyl betaine (e.g. lauroamidopropyl betaine); myristamidopropyl-, palm amidopropyl- or isostearylaminopropyl dimethylamine; sodium methyl cocoamide taurate or disodium methyl oleyl taurate; and the MONAQUATTM series (Mona Industries, Inc. , Paterson, N.J.); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (eg, pluronics, PF68, etc.); and the like. In certain embodiments, the surfactant is polysorbate 20.

As used herein, "treatment" refers to a clinical intervention that attempts to alter the natural history of the individual or cell being treated, and may be performed before or during a clinical pathological process. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease or its condition or symptom, alleviating the condition or symptom of the disease, attenuating any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or alleviating the disease state and alleviating or Improve prognosis.

An "effective amount" refers to an amount effective, at the dosage and for the time period required, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent may vary depending on factors such as the patient's disease state, age, sex and weight, and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

An "individual", "subject" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human and non-human primates) and rodents (eg, mice and rats). In certain embodiments, the mammal is a human.

An "agent" is an active drug used to treat a disease, disorder and/or condition.

"Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins with similar structural properties. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that generally lack antigen specificity. The latter type of polypeptides are, for example, produced at low levels by the lymphatic system and at high levels by myeloma.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies ( eg , full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies ( eg , Bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in more detail herein). Antibodies can be chimeric, human, humanized and/or affinity matured antibodies.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form that is not an antibody fragment as defined below. These terms especially refer to antibodies having heavy chains containing an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; bivalent antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of an antibody yields two identical antigen-binding fragments, termed "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, whose names reflect their ability to crystallize readily. Pepsin treatment produces F(ab') ₂ fragments that have two antigen combining sites and are still capable of cross-linking to antigen.

"Fv" is the smallest antibody fragment containing the entire antigen binding site. In a specific embodiment, a double-chain Fv species consists of a heavy chain and a light chain variable domain in a tight, non-covalently bound dimer. Collectively, the 6 CDRs of the Fv confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind an antigen, albeit with a lower affinity than the entire binding site.

The Fab fragment contains the heavy and light chain variable domains, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from one of the antibody hub regions. Fab'-SH refers to a Fab' in which the cysteine residue of the constant domain bears a free thiol group. F(ab') ₂ antibody fragments were originally produced as paired Fab' fragments with hinge cysteines. Other chemical couplings of antibody fragments are also known.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, and the individual antibodies comprising the population are identical except for possible mutations, such as natural mutations, which may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a mixture of different antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, a selection method may be to select a unique clone from a plurality of clones, such as a set of fusionoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence can be further altered to, for example, improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its in vivo immunogenicity, generate multispecificity Antibodies, etc., and antibodies comprising altered target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody system of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibody preparations is that they are generally free from contamination by other immunoglobulins.

The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be made by a variety of techniques, including, for example, the fusion tumor method (eg, Kohler et al., Nature , 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), Phage display technology (see, eg, Clackson et al., Nature , 352: 624-628 (1991); Marks et al., J. Mol. Biol . 222: 581-597 (1992); Sidhu et al., J. Mol. Biol . 338(2): 299-310 (2004); Lee et al., J. Mol. Biol . 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132 (2004), and use in animals to generate human immunoglobulin genes with sequences encoding human immunoglobulins technology for human or human-like antibodies to part or all of a locus or gene (see, eg, WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol . 7:33 (1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126 , No. 5,633,425, No. 5,661,016; Marks et al, Bio. Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-85 9 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol . 14: 845-851 (1996); Neuberger, Nature Biotechnol . 14: 826 (1996) and Lonberg and Huszar, Intern . Rev. Immunol . 13: 65-93 (1995).

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain is The remainder are identical or homologous to corresponding sequences of antibodies derived from another species or belonging to another class or subclass of antibodies, and fragments of such antibodies, so long as they exhibit the desired biological activity (US Pat. No. 4,816,567; and Morrison). et al, Proc. Natl. Acad. Sci. USA 81:6855-9855 (1984)).

"Native antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, an Ig native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variant heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variant light chain domain or light chain variable domain, followed by a light chain constant (CL) domain. Based on the amino acid sequences of the constant domains, the light chains of antibodies can be classified into one of two types called kappa and lambda.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody-antigen binding. The variable domains of the heavy and light chains of native antibodies ( _VH and _VL , respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) . ( See, eg , Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single _VH or _VL domain may be sufficient to confer antigen-binding specificity. In addition, _VH or _VL domains can be used to separate antibodies that bind a particular antigen from antibodies that bind that antigen to screen libraries of complementary _VL or _VH domains, respectively. See, eg , Portolano et al, J. Immunol. 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991).

A "humanized" antibody system refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will include substantially all of at least one (and usually two) variant domains, wherein all or substantially all HVRs (eg, CDRs) correspond to non-human antibodies, and the like, and all or Substantially all FRs correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the terms "hypervariable region", "HVR" or "HV" refer to regions of an antibody variable domain that are highly variable in sequence and/or form structurally defined loops. Typically, an antibody contains six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Many descriptions of hypervariable regions are in use and are encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Conversely, Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on analysis of available complex crystal structures. The residues of each of these HVRs are shown below. Ring Kabat AbM Chothia contacts L1/L24-L34; L24-L34/L26-L32/L30-L36: L2/L50-L56; L50-L56/L50-L52/L46-L55: L3/L89-L97; L89-L97 /L91-L96/L89-L96: H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

The hypervariable regions may include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 (L2) and 89- 97 (L3), and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) of VH. For each of these definitions, variable domain residues are numbered according to Kabat et al., supra.

Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several _of these can be further divided into subtypes (isotypes), such as _IgGi , IgG2, _IgG3 , _IgG4 , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described, eg, in Abbas et al., Cellular and Mol. Immunology , 4th ed. (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called a, 6, 8, y , and μ, respectively.

An "isolated" biomolecule (eg, nucleic acid, polypeptide or antibody) is one that has been identified and separated and/or recovered from at least one component of its natural environment.

"Subcutaneous administration device" refers to a device suitable or designed to administer a drug, such as a therapeutic antibody or pharmaceutical formulation, by the subcutaneous route. Exemplary hypodermic administration devices include, but are not limited to, needle safety devices (eg, those comprising prefilled syringes), injection devices (including auto-injectors, such as those comprising prefilled syringes), infusion pumps, injection pens, needle-free devices, and patches delivery system. A subcutaneous administration device can administer a volume of the pharmaceutical formulation, such as about 0.5 mL, about 0.7 mL, about 1.0 mL, about 1.25 mL, about 1.4 mL, about 1.5 mL, about 1.75 mL, about 2.0 mL, or about 5.0 mL .

"Package insert" or "marking" is used to refer to instructions usually included in commercial packaging of therapeutic products or agents containing indications, usage, dosage, administration, contraindications for the use of such therapeutic products or agents Symptoms, other therapeutic products and/or warnings in combination with packaged products.

As used herein, the term "gastrointestinal inflammatory disorders" refers to a group of chronic disorders that cause inflammation and/or ulceration in the mucosa. Such conditions include, for example, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis, indeterminate colitis, and infectious colitis), mucositis (eg, oral mucositis, gastrointestinal mucositis, nasal mucositis, and proctitis), necrotizing enterocolitis, and esophagitis.

"Inflammatory bowel disease" or "IBD" are used interchangeably herein and refer to bowel disease that causes inflammation and/or ulcers, and includes, but is not limited to, Crohn's disease and ulcerative colitis.

"Crohn's disease (CD)" or "ulcerative colitis (UC)" is a chronic inflammatory bowel disease of unknown etiology. Unlike ulcerative colitis, Crohn's disease can affect any part of the bowel. The most prominent feature of Crohn's disease is a granular, red-purple edematous thickening of the bowel wall. As inflammation progresses, these granulomas typically lose their circumscribed borders and fuse with surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. Like ulcerative colitis, the course of Crohn's disease can be continuous or recurrent, mild or severe, but unlike ulcerative colitis, Crohn's disease cannot be cured by resection of the affected bowel segment. Most people with Crohn's disease require surgery at some point, but then it usually recurs and often requires ongoing medical treatment.

Crohn's disease can involve any part of the digestive tract from the mouth to the anus, but it usually occurs in the ileocolonic, small bowel, or colon-anorectal regions. Histopathologically, the disease manifests as intermittent granulomas, crypt abscesses, dehiscence, and aphthous ulcers. The inflammatory infiltrate is a mixture of lymphocytes (T and B cells), plasma cells, macrophages, and neutrophils. A disproportionate increase in IgM- and IgG-secreting plasma cells, macrophages, and neutrophils occurred. The anti-inflammatory drugs sulfasalazine and 5-aminosalicylic acid (5-ASA) are used to treat mildly active colonic Crohn's disease and are often prescribed for maintenance of disease remission. Metroidazole and ciprofloxacin are similar in potency to sulfasalazine and appear to be particularly useful in the treatment of perianal disease. In more severe cases, corticosteroids are effective in treating worsening mobility and even maintaining remission. Azathioprine and 6-mercaptopurine have also been shown to be successful in patients requiring long-term administration of corticosteroids, and these drugs can also be used for long-term prophylaxis. Unfortunately, there can be extremely long delays (up to 6 months) before an effect occurs in some patients.

Antidiarrheal drugs may also provide symptomatic relief in some patients. Nutritional therapy or elemental diet can improve the nutritional status of patients and induce symptomatic improvement of acute disease, but it does not induce sustained clinical remission. Antibiotics are used to treat secondary intestinal bacterial overgrowth and to treat purulent complications.

"Ulcerative colitis (UC)" affects the large intestine. The course of the disease can be continuous or recurrent, mild or severe. The earliest lesions were inflammatory infiltrates with abscess formation at the base of the crypts of Lieberkühn. The combination of these distended and ruptured crypts often separates the overlying mucosa from its blood supply, resulting in ulceration. Symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent loose discharges that are mainly composed of blood, pus, and mucus with fewer fecal pellets. Acute, severe or chronic, nonremitting ulcerative colitis may require total colectomy. The clinical features of UC are highly variable, and onset may be insidious or sudden, and may include diarrhea, tenesmus, and recurrent rectal bleeding. Hirschsprung's disease, a life-threatening emergency, can occur with explosive involvement of the entire colon. Extraintestinal manifestations include arthritis, pyoderma gangrenosum, uveitis, and erythema nodosum.

The term "serum sample" refers to any serum sample obtained from an individual. Methods for obtaining serum from mammals are well known in the art.

The term "whole blood" refers to any whole blood sample obtained from an individual. Generally, whole blood contains all blood components, eg, cellular components and plasma. Methods for obtaining whole blood from mammals are well known in the art.

therapeutic agent

Provided herein are therapeutic agents for the treatment of inflammatory disorders of the gastrointestinal tract, such as inflammatory bowel disease. Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder that severely affects patients' quality of life and often requires surgical intervention (Casellas et al., Dig Dis . 1999;17(4):208–18; Carter et al., Gut . 2004; 53(Suppl 5):V1-16; Borren et al., Nat Rev Gastroenterol Hepatol . 2019;16(4):247-59). The major forms of IBD are ulcerative colitis (UC) and Crohn's disease, which are 2 distinct conditions that share some common symptoms and exhibit partially overlapping etiologies (Zhang and Li, World J Gastroenterol . 2014;20( 1):91–9; Abraham M and Cho, N Engl J Med . 2009;361(21):2066–78). Current pharmacological therapies for IBD are not curative. Additionally, many pharmacological therapies for IBD lose efficacy over the duration of the disease and can produce systemic side effects (AbrahaM & Cho, N Engl J Med . 2009;361(21):2066–78; Rogler, Best Pract Res Clin Gastroenterol . 2010;24(2):157–65). Irolizumab is an anti-β7 integrin monoclonal antibody developed for patients with UC and Crohn's disease. Irolizumab selectively inhibits α4β7 and αEβ7 to reduce the transport of immune cells to the gut and subsequent inflammatory effects on the gut lining (Zundler et al., Gut . 2019;68(9):1688-700). The efficacy and safety of ivolizumab in patients with UC was demonstrated in the Phase 2 EUCALYPTUS study (Vermeire et al., Lancet . 2014;384(9940):309-18).

In certain embodiments, the therapeutic agent is an anti-integrin beta7 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-integrin beta7 antibody is a humanized monoclonal anti-integrin beta7 antibody. In certain such embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs : HVR-H1, HVR-H2 and HVR-H3, wherein: (i) HVR-L1 comprises the amino acid sequence shown in SEQ ID NO: 1; (ii) HVR-L2 comprises the amino acid sequence shown in SEQ ID NO: 2 (iii) HVR-L3 comprises the amino acid sequence shown in SEQ ID NO: 3; (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO: 4; ( v) HVR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 5; and (vi) HVR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

In certain such embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof further comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain variable region , which comprises the amino acid sequence shown in SEQ ID NO:9. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain comprising SEQ ID The amino acid sequence shown in NO: 11; or a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region comprising SEQ ID The amino acid sequence shown in NO: 9.

In certain such embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof further comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: : the amino acid sequence shown in 11; or a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12. In certain such embodiments, the anti-integrin β7 antibody is ilrolizumab. SEQ ID NOs: 1-12 are provided below. Anti-integrin beta7 antibodies or antigen binding fragments thereof are further described in Intn'l Pub. No. WO2006/026759, which is incorporated herein by reference. serial identification number illustrate sequence 1 Anti-integrin beta7 antibody HVR-L1 RASESVDDDLLH 2 Anti-integrin beta7 antibody HVR-L2 KYASQSIS 3 Anti-integrin beta7 antibody HVR-L3 QQGNSLPNT 4 Anti-integrin beta7 antibody HVR-H1 GFFITNNYWG 5 Anti-integrin beta7 antibody HVR-H2 GYISYSGSTSYNPSLKS 6 Anti-integrin beta7 antibody HVR-H3.v1 RTGSSGYFDF 7 Anti-integrin beta7 antibody HVR-H3.v2 ARTGSSGYFDF 8 Anti-integrin beta7 antibody VL DIQMTQSPSSLSASVGDRVTITCRASESVDDLLHWYQQKPGKAPKLLIKYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSLPNTFGQGTKVEIKR 9 Anti-integrin beta7 antibody VH EVQLVESGGGLVQPGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQGTLVTVSS 10 Anti-Integrin β7 Antibody LC DIQMTQSPSS LSASVGDRVT ITCRASESVD DLLHWYQQKPGKAPKLLIKYASQSISGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ GNSLPNTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 11 Anti-integrin beta7 antibody HC.v1 EVQLVESGGG LVQPGGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQG TLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG 12 Anti-integrin beta7 antibody HC.v2 EVQLVESGGG LVQPGGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQG TLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

certain molecular biomarkers

In certain instances, biomarkers in biological samples obtained from subjects are quantified as a means of selecting subjects for treatment with a given therapeutic agent. International Patent Nos. WO2014160753, WO2015148809 and WO2009140684 describe methods of predicting the reactivity of a subject with an inflammatory disorder of the gastrointestinal tract to the anti-integrin beta7 antibody formulations described herein, and describe selection for use Methods of treating a subject having an inflammatory disorder of the gastrointestinal tract with the anti-integrin beta7 antibody formulations described herein.

General Techniques for Formulations

Formulations comprising anti-integrin beta7 antibodies or antigen-binding fragments thereof can be prepared and analyzed using certain excipients and techniques known in the art and as further described herein. In certain embodiments, the antibody to be formulated has not been subjected to prior lyophilization, and the formulations of interest herein are aqueous formulations. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody in the formulation is an antibody fragment, such as F(ab') ₂ , in which case it may be necessary to address issues that may not arise for full-length antibodies (eg, antibody trimming to Fab) . The therapeutically effective amount of antibody present in the formulation is determined by taking into account the desired dose volume and mode of administration. In certain embodiments, about 0.1 mg/mL to about 250 mg/mL or about 10 mg/mL to about 220 mg/mL or about 50 mg/mL to about 220 mg/mL or about 100 mg/mL to about 220 mg/mL or about 100 mg/mL to about 150 mg/mL or about 150 mg/mL to about 200 mg/mL are exemplary antibody concentrations in the formulation. In certain embodiments, the anti-integrin beta7 antibody is formulated at a concentration of 150 mg/mL.

Aqueous formulations of anti-integrin beta7 antibody or antigen-binding fragment thereof in pH-buffered solution are prepared. The buffer may have a pH in the range of about 4.5 to about 6.5. In certain embodiments, the pH is greater than 5.0 and up to 7.0. In certain embodiments, the pH is greater than 5.5. In certain embodiments, the pH is between 5.5 and 6.1. In certain embodiments, the pH is between 5.6 and 6.1. In certain embodiments, the pH is at or about 5.8. In certain embodiments, the pH is 5.8. In certain embodiments, the pH is between 5.7 and 5.9. In certain embodiments, the pH is between 5.75 and 5.85. The pH of the formulations disclosed herein are higher than standard values for antibody formulations with similar excipient compositions. A typical antibody formulation has a pH of 5.5, while the formulations disclosed herein have a pH greater than 5.5, such as a pH of 5.8, between 5.7 and 5.9, or between 5.75 and 5.85. Higher formulation pH reduces the risk of particle formation due to polysorbate degradation during long-term storage in prefilled syringes at high protein concentrations. The reduced risk of particle formation is due to the increased solubility of free fatty acids available from polysorbate degradation at higher pH. A pH greater than 5.5 (eg pH 5.8) balances the risk of particle formation with the chemical and physical stability of the antibody. A pH greater than 5.5 (eg pH 5.8) will minimize the rate of Asp isomerization and formation of succinimide intermediates, thereby not substantially affecting the chemical stability of the antibody and thus the product by allowing storage at ambient temperature quality to make it easy for the patient to use the device.

Examples of buffers that control the pH within this range include acetates (eg histidine acetate, arginine acetate, sodium acetate), succinates (eg histidine succinate, arginine succinate) salt, sodium succinate), gluconate, citrate and other organic acid buffers and combinations thereof. Depending on, for example, the buffer and the desired isotonicity of the formulation, the buffer concentration can range from about 1 mM to about 600 mM. In certain embodiments, the buffer contains histidine. The presence of histidine in the formulation can greatly reduce the rate of formation of high molecular weight species (HMWS) in the presence of zinc. The concentration of histidine in the formulation can be between about 5 mM and about 40 mM, between about 5 mM and about 30 mM, between about 10 mM and about 40 mM, between about 10 mM Between mM and about 30 mM, between about 15 mM and about 25 mM, between about 10 mM and about 20 mM, or between about 15 mM and about 20 mM. In certain embodiments, the concentration of histidine in the formulation is about 20 mM.

In certain embodiments, the buffer is 20 mM histidine (pH 5.8).

In certain embodiments, the formulation comprises arginine succinate. In certain embodiments, the concentration of arginine succinate in the formulation is about 20 mM to 300 mM. In certain embodiments, the salt concentration of arginine succinate in the formulation is about 100 mM to 300 mM, about 100 mM to about 200 mM, about 150 mM to about 300 mM, about 200 mM to about 300 mM , about 100 mM to about 250 mM, about 150 mM to about 250 mM, or about 150 mM to about 200 mM. In certain embodiments, the concentration of arginine succinate in the formulation is about 200 mM. The high arginine concentration and high conductivity formulations shield the charge on the antibody and prevent pH changes. In addition, arginine can affect the viscosity of the formulation, for example, the viscosity of the formulation is reduced by the addition of arginine to the formulation.

The formulation has a viscosity of less than about 20 centipoise (cP) at 25°C. In certain embodiments, the formulation has a viscosity between about 1 cP and about 20 cP at 25°C, between about 5 cP and about 20 cP at 25°C, and a viscosity at 25°C between Between about 5 cP and about 15 cP, a viscosity at 25°C between about 1 cP and about 10 cP, or a viscosity at 25°C between about 5 cP and about 10 cP. In certain embodiments, the formulation has a viscosity of about 7 cP at 25°C .

In certain embodiments, the antibody formulation includes a surfactant. Exemplary surfactants include nonionic surfactants. Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), Pronic polyols, Triton®, polyoxyethylene sorbitan Monoethers (Tween®-20, Tween®-80, etc.), lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride. In certain embodiments, the surfactant is polysorbate 20.

Non-ionic surfactants can help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without denaturing the active therapeutic protein or antibody.

The amount of surfactant added should be such that aggregation of the formulated antibody is reduced and/or particle formation in the formulation is minimized and/or adsorption is reduced. For example, the surfactant can be present in the formulation in an amount greater than 0.005% weight/volume (w/v). In certain embodiments, the concentration of surfactant in the formulation is greater than 0.005% w/v and up to about 1% w/v. The concentration of surfactant in the formulation can be between about 0.005% w/v and about 0.5% w/v, between about 0.02% w/v and about 0.5% w/v, between about 0.03% w/v Between % w/v and about 0.5% w/v, between 0.03% w/v and 0.1% w/v. In certain embodiments, the concentration of surfactant in the formulation is 0.04% w/v. In certain embodiments, the surfactant is polysorbate 20 present in the formulation in an amount of 0.04% w/v. A typical concentration of polysorbate 20 for antibody formulations is 0.02% (w/v). The formulations disclosed herein contain 0.04% w/v polysorbate 20. Higher concentrations of polysorbate 20 help dissolve free fatty acids that can be formed from polysorbate degradation, thereby reducing the risk of particle formation.

In certain embodiments, the formulations contain the above-described agents (eg, antibodies, buffers, and surfactants) and are substantially free of one or more preservatives (eg, benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium chloride). In certain embodiments, the formulations contain no preservatives. In certain embodiments, a preservative may be included in the formulation, especially where the formulation is a multiple dose formulation. Concentrations of preservatives may range from about 0.1% to about 2% or from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients, or stabilizers (such as those described in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. ed. (1980)) may be included in the formulation, provided that they The desired properties of the formulation are not adversely affected. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include other buffers; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; Metal complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; and/or salt-forming counter ions.

Although the various descriptions of chelating agents herein generally focus on EDTA, it should be understood that other metal ion chelating agents are also encompassed by the present invention. Metal ion chelators are well known to those skilled in the art and include, but are not necessarily limited to, amine polycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid), NTA (nitrotriacetic acid), EDDS (ethylenediaminedisuccinate), PDTA (1,3-propanediaminetetraacetic acid), DTPA (diethylene diethylene triaminepentaacetic acid), ADA (beta-alanine diacetic acid), MGCA (methylglycine diacetic acid), etc. Additionally, some embodiments herein include phosphonate/phosphonic acid chelating agents.

Tonicity agents (sometimes referred to as "stabilizers") are present to adjust or maintain the tonicity of liquid compositions. When used with larger, charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged groups on amino acid side chains, thereby reducing the size of the molecule potential for intermolecular and intramolecular interactions. The tonicity agent may be present in any amount between 0.1% to 25% by weight or 1% to 5%, taking into account the relative amounts of the other ingredients. Tonicity agents include polyhydric, trihydric or more polyhydric sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Other stabilizers include a wide range of excipients whose functions range from bulk builders to solubility enhancers and up to agents that prevent denaturation or sticking to the container walls. Stabilizers may be present in the range of 0.1 - 10,000 parts/weight active protein or antibody. Typical stabilizers include: polyalcohols (listed above); amino acids such as alanine, glycine, glutamic acid, aspartic acid, histidine, arginine, lysine, Methionine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, Mannose, sorbose, xylose, ribose, ribitol, inositol, inositol, galactose, galactitol, glycerol, cyclic polyols (eg, inositol), polyethylene glycol; sulfur-containing reducing agents such as urea , glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or others Immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (eg xylose, mannose, fructose, glucose; disaccharides (eg lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Various analytical techniques for measuring protein stability are available in the industry and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, ed. Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 2990 (1993). Stability can be measured at selected temperatures and over selected time periods. In certain embodiments, the formulations are stable at about 40°C for at least about 1 week. In certain embodiments, the formulations are stable at about 5°C for at least about 12 months, and/or at about 5°C for at least about 18 months, and/or at about 5°C for at least about 2 years, and/or stable at about 5°C for at least about 3 years, and/or stable at about 5°C for at least about 4 years, and/or stable at about 5°C for at least about 5 years. In certain embodiments, the formulations are stable at about -20°C for at least 2 years, and/or at about -20°C for at least 4 years, and/or at about -20°C for at least about 5 years, and and/or stable at about -20°C for at least about 6 years, and/or stable at about -20°C for at least about 7 years. In certain embodiments, the formulations are stable at about 25°C for at least about 1 week, and/or at about 25°C for at least about 2 weeks, or at about 25°C for at least about 4 weeks. In certain embodiments, the formulation remains stable after freezing (to, eg, -70°C) and thawing the formulation, eg, after 1, 2, 3, 4, or 5 freeze-thaw cycles. Stability can be assessed qualitatively and/or quantitatively in a variety of different ways, including assessing aggregate formation (eg, using particle size sieve chromatography, by measuring turbidity, and/or by visual inspection); by using cation exchange chromatography , imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis to assess charge heterogeneity; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptides Graph (eg, trypsin or LYS-C) analysis; assess the biological activity or antigen-binding function of antibodies; and the like. Instability can involve any one or more of the following: aggregation, deamidation (eg Asn deamidation), oxidation (eg Met oxidation), isomerization (eg Asp isomerization), Trimming/hydrolysis/fragmentation (eg hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal manipulation, differential glycosylation, etc.

Formulations for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes before or after preparation of the formulation.

In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is administered using, for example, a self-injection device, an auto-injector device, or other device designed for self-administration. In certain embodiments, the anti-integrin beta7 antibody or antigen-binding fragment thereof is administered using a subcutaneous administration device. Various self-injection devices and subcutaneous administration devices, including auto-injector devices, are known in the art and are commercially available. Exemplary devices include, but are not limited to, prefilled syringes (eg, BD HYPAK SCF®, BD NEOPAK™, READYFILL ^™ , and STERIFILL SCF ^™ from Becton Dickinson; CLEARSHOT ^™ copolymer prefilled syringes from Baxter; and available from West Pharmaceutical Services Daikyo Seiko CRYSTAL ZENITH® Pre-Filled Syringe); single-use pen injection devices such as the BD Pen from Becton Dickinson; ultra-sharp and micro-needle devices such as the INJECT-EASE ^Tm and Micro Infuser devices from Becton Dickinson; and H-PATCH ^™ ) from Valeritas and needle-free injection devices (eg, BIOJECTOR® and IJECT®, available from Bioject; and SOF-SERTER® patch devices, available from Medtronic). Certain embodiments of subcutaneous administration devices are further described herein. The use of such self-injection or subcutaneous delivery devices is contemplated for co-formulation or co-administration of an anti-integrin beta7 antibody or antigen-binding fragment thereof and at least a second therapeutic compound.

Recombination method

Antibodies can be produced using recombinant methods and compositions, eg, as described in US Pat. No. 4,816,567. In certain embodiments, nucleic acid molecules encoding the anti-integrin beta7 antibodies or antigen-binding fragments thereof described herein are provided. The nucleic acid molecule can encode an amino acid sequence comprising _VL and/or an amino acid sequence comprising the _VH of an antibody (eg, the light and/or heavy chain of an antibody). In certain embodiments, one or more vectors (eg, expression vectors) comprising the nucleic acid molecules are provided. In certain embodiments, host cells comprising the nucleic acid molecule are provided. In certain embodiments, the host cell comprises (eg, has been transformed): (1) a vector comprising a nucleic acid molecule encoding the amino acid sequence comprising the _VL of the antibody and the amino acid sequence comprising the _VH of the antibody; or ( 2) a first vector comprising a nucleic acid molecule encoding the amino acid sequence comprising the _VL of the antibody; and a second vector comprising a nucleic acid molecule encoding the amino acid sequence comprising the _VH of the antibody. In certain embodiments, the host cells are eukaryotic cells, eg, Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells). In certain embodiments, a method of making an anti-integrin beta7 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid molecule encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally from The host cell (or host cell culture medium) recovers the antibody.

In recombinant production of anti-integrin beta7 antibodies, nucleic acid molecules, such as those encoding the antibodies described above, are isolated and inserted into one or more vectors for further colonization and/or expression in host cells. These nucleic acid molecules can be readily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially without glycosylation and Fc effector functions. For the expression of antibody fragments and polypeptides in bacteria, see, eg, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (Edited by B.K.C. Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression , the polypeptide can be separated from the soluble fraction in bacterial cell paste and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable hosts for cloning or expression of antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", thereby Resulting in the production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (discussing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines are: the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney line (as described in Graham et al., J. Gen Virol. 36:59 (1977) ) 293 or 293 cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1) ; African green monkey kidney cells (VERO-76); Human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); G2); mouse mammary tumor (MMT 060562); TRI cells, as described in Mather et al., Annals N.Y.Acad. Sci. 383: 44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, For example YO, NSO and Sp2/0. For a review of some suitable mammalian host cell lines for antibody production see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (Edited by B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003 ).

check

The anti-integrin beta7 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

The evolizumab efficacy assay measures the ability of evolizumab to inhibit the binding of RPMI8866 B cells to MAdCAM. In this assay, MAdCAM is coated on a 96-well microtiter plate. After an overnight incubation, ibolizumab standards, controls and samples, and a fixed amount of cells were added to the plates. Plates were incubated at 37°C in a humidified incubator to allow binding of cells to MAdCAM. A washing step was performed to remove non-adherent cells and the remaining viable cells were quantified by adding the redox dye alamar Blue, which is blue and non-fluorescent in its oxidized state but reduced by the intracellular environment In a highly fluorescent pink form. Therefore, the change in color and fluorescence is proportional to the number of bound living cells. Results are expressed in RFU and plotted against ivolizumab concentration. Parallel curve analysis was used to estimate the activity of the elezolizumab samples relative to the reference material.

Efficacy test

Products and Kits

An article of manufacture is provided comprising the formulation and instructions for its use are provided. The article of manufacture contains a container.

In certain embodiments, there is provided an article of manufacture comprising a subcutaneous administration device that delivers to a subject a fixed dose of an anti-integrin beta7 antibody or antigen-binding fragment thereof, wherein the fixed dose is, for example, but not limited to, 105 mg or 210 mg . In certain embodiments, the anti-integrin beta7 antibody is ilozumab. Formulating an anti-integrin beta7 antibody or antigen-binding fragment thereof in a subcutaneous administration device in a buffer (eg, pH 5.8 histidine) and other excipients (eg, polysorbate and arginine succinate), , thereby being provided as a stable pharmaceutical formulation.

In certain embodiments, the hypodermic delivery device is a prefilled syringe comprising a glass barrel having a needle and optionally a needle shield and optionally a needle shield device. In certain embodiments, the volume of the formulation contained in the syringe is between about 0.1 mL and about 2 mL, between about 0.1 mL and about 2 mL, between about 0.5 mL and about 2 mL Occasionally between about 1 mL and about 2 mL. In certain embodiments, the volume of the formulation contained in the syringe is between about 0.5 mL and about 2 mL. In certain embodiments, the volume of the formulation contained in the syringe is about 0.5 mL, about 0.7 mL, about 1 mL, about 1.4 mL, about 1.5 mL, or about 2.0 mL. In certain embodiments, the volume of the formulation contained in the syringe is about 0.7 mL. In certain embodiments, the volume of the formulation contained in the syringe is about 0.75 mL. In certain embodiments, the volume of the formulation contained in the syringe is about 1 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is between about 0.5 mL and about 1.0 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is between about 1.0 mL and about 1.5 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.4 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.5 mL. In certain embodiments, the volume of the formulation contained in the prefilled syringe is about 1.45 mL.

In certain embodiments, the needle is a dropper needle comprising a 3-bevel tip or a 5-bevel tip. In certain embodiments, the needle is between 25 gauge (G) and 30G. In certain embodiments, the needle is between 1/2 inch long and 5/8 inch long. In certain embodiments, the hypodermic administration device comprises a prefilled 1.0 mL low tungsten borosilicate glass (Type I) syringe and stainless steel 5-bevel 27G 1/2 inch long thin walled insertion needle. In certain embodiments, the subcutaneous administration device comprises a rigid needle shield. In certain embodiments, the rigid needle shield includes a rubber formulation with low zinc content and includes a rigid polypropylene shield. In certain embodiments, the rubber plunger comprises Daikyo 777-7 rubber with a FluroTec® ethylene-tetrafluoroethylene (ETFE) coating (West Pharmaceutical Services, Inc.). In certain embodiments, the subcutaneous administration device includes a needle safety device. Exemplary needle safety devices include, but are not limited to, Ultrasafe Passive® Needle Guard X100L (Becton Dickinson and Company) and Rexam Safe n Sound ^™ (Rexam).

In certain embodiments, the injection device is a prefilled syringe. Non-limiting examples of prefilled syringes include BD HYPAK SCF®, READYFILL ^™ , and STERIFILL SCF ^™ from Becton Dickinson; CLEARSHOT ^™ copolymer prefilled syringes from Baxter; and Daikyo Seiko CRYSTAL ZENITH® prefilled syringes available from West Pharmaceutical Services. In certain embodiments, the prefilled syringe contains silicone oil. Development of a prefilled syringe of elezolizumab with optimal silicone oil content to ensure low injection force while maintaining a low risk of particle formation. Depending on pH and polysorbate concentration, free fatty acids from degraded polysorbate are distributed more or less into the polysiloxane oil. Thus, given the combination of target pH and target silicone oil content, the particular combination of formulation excipients is well suited for long-term storage of high concentration antibody formulations stored in prefilled syringes. In certain embodiments, the amount of silicone oil in the prefilled syringe is no greater than about 1 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is between about 0.1 mg and about 1 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is between about 0.2 mg and about 0.6 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is between about 0.5 mg and 0.9 mg.

The prefilled syringe can have any suitable syringe capacity. In certain embodiments, the syringe volume of the prefilled syringe is between about 0.5 mL and about 10 mL, between about 0.5 mL and about 5 mL, between about 0.5 mL and about 2.5 mL, between about 0.5 mL and about 2.5 mL. Between about 1 mL and about 5 mL or between about 1 mL and about 2.5 mL. In certain embodiments, the prefilled syringe has a syringe capacity of 1 mL. In certain embodiments, the prefilled syringe has a syringe capacity of 2.25 mL.

In certain embodiments, the injection device is an auto-injector, such as the auto-injectors disclosed in US Patent Nos. 2014/0148763 and 2014/0114247, which are incorporated herein by reference. In certain embodiments, the auto-injector is a single-use auto-injector. In certain embodiments, the auto-injector is based on ^Rotaject® technology (eg, available at https://www.shl.group/products-and-services/rotaject-technology-auto-injector/).

Other devices suitable for subcutaneous delivery include, for example, but not limited to, injection devices such as INJECT-EASE ^™ and GENJECT ^™ devices; infusion pumps such as ACCU-CHECK ^™ ; injection pens such as BD Vystra™ from Becton Dickinson; needle-free devices , such as MEDDCTOR ^™ and BIOJECTOR ^™ and IJECT ^™ ; and subcutaneous patch delivery systems such as BD Libertas™, H-PATCH ^™ (available from Valeritas) and SOF-SERTER ^™ .

Kits typically contain the above-mentioned containers and one or more other containers containing materials desired from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and packaging with instructions for use insert. Indicia may be present on the container to indicate that the composition can be used for a particular therapy.

Example

The following are examples of formulations and methods of the present invention. It should be understood that various other embodiments may be practiced in light of the general description given above.

example 1

Important aspects in optimizing the formulation and configuration of the evolizumab drug product are minimizing viscosity to minimize injection force, maximizing accuracy of injection volume, and optimizing compatibility with device components. It is important to balance the optimal formulation excipients to adequately protect the antibody from physical and chemical degradation, while maintaining biological activity (efficacy) and achieving high antibody concentrations, while maintaining low viscosity. These aspects of the formulation must be coordinated with the configuration and specifications of the prefilled syringe (eg, silicone oil concentration, syringe construction material, barrel width, and needle wall thickness) to achieve lower injection forces, shorter injection times, and better performance. Small injection source pain.

In the present example, preparations comprising 150 mg/mL irolizumab, 20 mM histidine, 200 mM arginine succinate and 0.04% w/v polysorbate 20 (PS20) at pH 5.8 formulations of irolizumab. This formulation of irolizumab has been shown to be surprisingly suitable for long-term storage at high concentrations in prefilled syringes. Additionally, when paired with prefilled syringes, this formulation minimizes the risk of sub-visible and visible particle formation during long-term storage, thereby extending shelf life and achieving patient convenience. To study the effect of formulation pH, arginine, succinate, polysorbate 20, antibody concentration, and histidine on the viscosity and stability (eg, aggregate formation, charge variants, etc.) of ivolizumab formulations .

pH and polysorbate 20

The effect of polysorbate 20 and pH of the solution (formulation buffer) on the solubility of lauric, myristic, and palmitic acids was investigated. As shown in Figure 1, increasing the level of polysorbate 20 increases the solubility of lauric acid, myristic acid, and palmitic acid (which can be generated from the degradation of polysorbate), thereby reducing the risk of particle formation. The results presented in Figure 1 also show that increasing pH increases the solubility of free fatty acids. pH 5.8 is the optimal formulation pH, which balances the risk of particle formation with the chemical and physical stability of the antibody. Formulations with this pH can minimize the rate of aspartic acid (Asp) isomerization and succinimidium intermediate formation, thereby not substantially affecting the chemical stability of the antibody and by allowing storage at ambient temperature. This product quality makes it easy for the patient to use the device in combination. In addition, the reduced risk of particle formation is due to the increased solubility of free fatty acids available from polysorbate degradation at higher pH.

Arginine

Lower viscosity is desirable because low viscosity reduces injection force and ensures accuracy of injection volume. The use of arginine hydrochloride or arginine succinate in antibody formulations has been described previously. See, for example, US Patent No. 8,142,776 and International Patent Application Publication Nos. WO2006065746 and WO2010102241. To investigate the effect of arginine succinate on the viscosity of formulations. First, the viscosities of the formulations of elezolizumab with arginine succinate were compared to those of the formulations of ilrolizumab without the presence of arginine succinate, and the results are shown in Figure 2. As shown in Figure 2, the solution viscosity was significantly reduced by the addition of arginine succinate to the formulation. Next, the inventors investigated whether arginine, in this case 200 mM arginine succinate, could affect the viscosity of irolizumab formulations with different antibody concentrations. The viscosities of formulations containing ilrolizumab at 100 mg/mL, 150 mg/mL, 180 mg/mL, 200 mg/mL, and 220 mg/mL were measured and the results are shown in Figure 3. As shown in Figure 3, with arginine succinate (eg, 200 mM arginine succinate), formulations with antibody concentrations up to 200 mg/mL have suitable pre-filled syringe (PFS) doses. given viscosity.

Silicone oil

Because pre-filled syringe pharmaceutical products often contain silicone oils and because silicone oils are known to cause protein aggregation and/or particle formation over time, the inventors investigated the effect of silicone oils on ilozumab formulations.

To test the effect of silicone oil, silicone oil was sprayed into the interior of the glass barrel of a prefilled syringe prior to filling the ellozumab formulation. To evaluate protein aggregation, particle size sieve chromatography (SEC) was used to determine the high molecular weight species (HMWS) of elezolizumab to determine the aggregation and oligomeric state of the antibodies (including HMWS) in the formulations of elezolizumab . SEC was performed on an Agilent High Performance Liquid Chromatography (HPLC) system using a Tosoh TSKgel column and a mobile phase consisting of potassium phosphate and potassium chloride at a flow rate of 0.5 mL/min and quantified by UV absorbance and peak area integration . Briefly, the effect of various amounts of polysiloxane oil on HMWS formation was determined in formulations containing elezolizumab at 150 mg/mL or 180 mg/mL. As shown in Figure 4, increasing polysiloxane oil content did not affect the physical stability of all tested ilrolizumab formulations under various conditions.

Therefore, the specific combination of formulation excipients at the target pH and in the presence of silicone oil is well suited for long-term storage of high concentration antibody formulations stored in prefilled syringes. This finding is corroborated by the following results: Irolizumab has long-term stability in prefilled syringes for approximately 6 years (eg, 74 months), and no visible particles are observed.

Succinate and histidine

The formulations disclosed herein have the advantage of protecting the antibody from additional stress that may result from storage in prefilled syringes. Leachables from prefilled syringes can cause chemical and physical degradation of antibodies. For example, zinc and tungsten can contribute to metal-catalyzed degradation. During the syringe manufacturing process, a hot tungsten pin was inserted into the glass barrel to create a hole for needle insertion. This process can leave residual tungsten particles in the syringe barrel, which can interact with the drug solution and cause aggregates to form. Tungsten induces protein aggregation and the formation of proteinaceous particles. Tungsten can also induce protein oxidation, resulting in protein aggregation. Succinic acid prevents zinc-based metal-catalyzed degradation. In addition, the formulations disclosed herein are not susceptible to tungsten doping mediated aggregation.

To determine the effect of tungsten exposure on the physical stability of the ilrolizumab formulations, the percent HMWS over time was determined after exposure to various amounts of tungsten. As shown in Figure 5, tungsten did not affect the product quality of irolizumab.

Both the plunger and the needle shield may be constructed of a rubber material that allows zinc to leach into the formulation. It is well known that zinc can complex with proteins to create protein aggregates (eg HMWS) and increase the viscosity of antibody formulations. The effect of zinc on the viscosity of ivolizumab formulations at different protein concentrations was investigated. As shown in Figure 6, the concentration of ilozumab in formulations containing 20 mM histidine, 200 mM arginine succinate (pH 5.8) was 150 mg/mL or less, formulated without zinc There was no difference in viscosity between the formulation and the formulation containing 10 mM zinc. Zinc increases viscosity at irolizumab concentrations above 150 mg/mL. In addition, Figure 7 shows the formation of protein aggregates in formulations containing 150 mg/mL of irolizumab and 50 mM zinc. Figure 8 shows that the addition of zinc increased the percentage of HMWS and that the increase in the percentage of HMWS at 50 mg/mL evolizumab was higher than that at 10 mg/mL evolizumab.

The inventors then tested whether histidine could reduce the formation of HMWS. As shown in Figure 9, the presence of histidine in the formulation greatly reduced the rate of HMWS formation in the presence of zinc. In addition, as shown in Figure 10, succinate also inhibited the HMWS-forming interaction between ivolizumab and zinc. Next, the effect of different concentrations of histidine and succinate on the formation of HMWS was investigated. As shown in Figure 11, the combination of histidine and succinate minimizes HMWS formation in the presence of zinc.

Drug Substances and Drug Substance Stability

Ebolizumab drug formulated at 150 mg/mL in 20 mM histidine (pH 5.8), 200 mM arginine succinate, 0.04% polysorbate 20 was evaluated at 30°C, 5°C, and -20°C Stability of Substances (DS). Stability was also assessed for up to 5 freeze/thaw cycles to support large-scale storage and handling. No changes in chemical, physical or biological activity properties were observed after 5 freeze/thaw cycles or after DS was stored at -20°C for 7 years (84 months). After 6 months of storage at 5°C, no change was observed by all assays except ion exchange chromatography. Degradation was measured by non-reducing capillary electrophoresis, ion exchange and particle size sieve chromatography after 14 days of storage at 30°C.

Deamidation and isomerization are degradation pathways that can occur in terms of stability. Ion exchange chromatography (IEC) was used to monitor chemical degradation and quantify acidic variants, basic variants and main peaks. No changes in main peaks, acidic variants or basic variants were observed after 5 freeze/thaw cycles or after 84 months of storage at -20°C. After 6 months of storage at 5°C, a 1.7% loss of the main peak was observed along with an increase of 1.0% and 0.8% for the acidic and basic variants, respectively. After 14 days of storage at 30°C, the main peak lost 4.5% and the acidic and basic variants increased by 2.1% and 2.4%, respectively. Qualitatively, no change in ion exchange profile or new peaks was observed.

After 14 days of storage at 30°C, the following changes were observed: a loss of 1.0% in the main peak and a simultaneous increase of 0.82% and 0.14% in the front and back peaks, respectively, as observed by non-reducing capillary electrophoresis-SDS; the main peak was observed by IEC Loss of 4.5% and concurrent increases of 2.1% and 2.4% for acidic and basic variants; 0.28% and 0.09% for HMWS and LMWS, respectively, as observed by particle size sieve chromatography (SEC).

The study investigated the drug product of irolizumab formulated at 150 mg/mL in 20 mM histidine (pH 5.8), 200 mM arginine succinate, 0.04% polysorbate 20 in a 105 mg (0.7 ml) prefilled syringe group stability in the state. Irolizumab showed no change in product quality after 60 months at 5°C, as determined by clarity, opalescence and coloration (COC), pH, protein concentration, size sieve chromatography (SEC) and efficacy. Evaluation. A 7.7% loss of the main peak was found by ion exchange chromatography (IEC) with a simultaneous increase of 4.2% and 3.4% for the acidic and basic variants, respectively, and a 1.0% loss of the main peak by non-reducing CE-SDS. No change in clarity, opalescence and coloration (COC), pH, protein concentration or efficacy after 3 months storage at 25°C; 0.4% loss of main peak and high and low molecular weight species by particle size sieve chromatography Substances were all increased by 0.2%; the main peak was found to lose 1.3% by CE-SDS; the main peak was found to be 9.8% lost by ion exchange chromatography and increased by 5.1% for acidic variants and 4.6% for basic variants. Irolizumab drug products stored at 5°C for 60 months contain virtually no visible particles.

in conclusion

A unique aspect of the specific excipient combination of the elezolizumab formulation can protect the antibody from additional stress that can result from storage of the antibody in a prefilled syringe, for example, histidine and succinate can reduce the amount of HMWS formed by zinc and antibodies. A typical antibody formulation contains 0.02% w/v polysorbate 20 and a typical antibody formulation is at pH 5.5. Higher than typical polysorbate 20 concentrations (eg, 0.04% w/v) and higher than typical pH (eg, pH 5.8) dissolve free fatty acids, thereby reducing the risk of particle formation from polysorbate degradation, such as in During long-term storage in prefilled syringes. The combination of excipients for the formulation of ilvolizumab can be particularly effective in controlling pH during tangential flow filtration (TFF) in large scale manufacturing. The high arginine concentration and high conductivity formulations shield the charge on the antibody and prevent pH changes. The histidine concentration can effectively buffer the formulation at the target pH. This is important to ensure stable pH control during manufacturing and to have formulations with a narrow pH range to enable this optimal configuration. The formulations also maintain chemical and physical stability and maintain efficacy over different time periods and at different temperatures as described above.

example 2 - A clinical strategy and initial results to support the use of evolizumab auto-injectors in healthy volunteers

SUMMARY OF THE INVENTION

Irolizumab is a novel, dual-acting, anti-beta7 integrin antibody being developed for patients with moderate to severe ulcerative colitis or Crohn's disease. The Phase 3 study utilized a prefilled syringe (PFS) to administer irolizumab. At the same time, auto-injectors (AI) are being developed to increase delivery options for patients. This example illustrates the overall R&D strategy and details this first human study of R&D AI.

This open-label study in healthy volunteers (HVs) evaluated the tolerability and usability of the developed ilrolizumab AI. The primary endpoint was the proportion of participants with greater than mild pain after injection. Adverse events (AEs) and usage errors were also evaluated. Results are reported according to injection site (thigh vs. abdomen) and needle training (experienced vs. non-experienced). Pharmacokinetic (PK) variability between participants was included as an exploratory endpoint.

30 participants completed the study. A total of 97% of participants never experienced more than mild pain during the study; 50% experienced no pain at all. Three usage errors were observed, one of which resulted in partial dose delivery of ivolizumab. No pattern of usage error was observed. A mild injection site reaction (ISR) was reported; it resolved by the end of the study. Participants injected into the abdomen reported more ISR than those injected into the thigh; needle training did not appear to affect the incidence or severity of AEs.

Results from this first-in-human study confirmed that a single injection of 105 mg of elezolizumab using AI was well tolerated in HV with transient, mild pain and minimal use error. The results of this study also inform the design of subsequent PK comparability studies comparing PFS and AI. All in all, the availability of AI could provide an attractive option for patients who expect a convenient, easy-to-use delivery mechanism for ivolizumab.

Introduction

Irolizumab is being evaluated in an extensive clinical program in a Phase 3 study in patients with moderate to severe UC and Crohn's disease (Etro Studies. The Etro Studies: Explore Innovation: Contribute to Science. Genentech; 2019. July 2019 26), in which irolizumab was administered once a month via subcutaneous (SC) injection using a prefilled syringe (PFS) with a needle safety device (NSD).

Disposable, prefilled auto-injectors (AI) have a number of potential advantages over PFS-NSDs; most notably, their ability to keep the needle out of sight of the user at all times during the injection. Example PFS and AI are shown in FIG. 12 . AI also increases convenience, ease of use, reduces the risk of dose errors, and improves patient comfort. Studies consistently show that many self-administered patients prefer AI over syringe-based devices (Kivitz et al, Clin Ther . 2006;28(10):1619-29; Kivitz and Segurado, Expert Rev Med Devices . 2007;4(2 ): 109-16; Borras-Blasco et al., Expert Opin Biol Ther. 2010;10(3):301-7; Vermeire et al., Patient Prefer Adherence . 2018;12:1193-202). For example, a recent study of golimumab in UC patients confirmed that most patients prefer AI administration over PFS due to increased ease of use and reduced injection discomfort ( Vermeire et al, Patient Prefer Adherence . 2018;12:1193–202).

The AI currently under development consists of an automated delivery system that encapsulates the same PFS used in the Phase 3 study ( see Figure 13). The drug product contained in the AI and PFS-NSD consisted of a liquid formulation of a 105 mg solution of ivolizumab (0.7 mL, nominal volume 150 mg/mL) for single-dose administration. The entire dose is usually administered for about 2 seconds.

AI includes a number of features aimed at improving the patient experience and increasing patient self-administration comfort. The automated drug delivery system is activated by lightly pressing the device vertically against the skin. Upon activation, the AI automatically inserts the needle and dispenses the syringe contents. After the injection is complete, the needle cover extends and locks the needle, keeping the needle out of sight at all times during the injection and protecting the user and others from accidental contact with the needle in use. The AI also incorporates visual and auditory mechanisms designed to help the user self-inject; namely, a visual gyroscope and an audible click, both of which indicate that drug administration is in progress or complete. Additionally, the vision rod moves across the viewing window while the injection is in progress.

Here, the overall development strategy for a novel AI for elezolizumab and the findings of the first-in-human study of this device are presented. The primary objectives of this study (NCT02629744) were to evaluate the safety and tolerability of ivolizumab administered by the AI and primarily at injection site pain after self-injection with the AI and to document critical usage errors. Additionally, the inventors evaluated this relatively unique study design using a combination of error assessments (traditionally conducted as simulation studies) with tolerability, safety, and exploratory PK studies.

method

Study Design and Procedures . This first-in-human AI tolerance study is an open-label, single-arm study in healthy volunteers evaluating pain, safety, and AI availability upon subcutaneous self-administration. Assign participants (1:1) into 2 groups. To simulate prior experience with self-injection, 1 group ("Needle Experienced") was trained prior to self-injection using the AI; the other group ("Needle Experienced") did not. The training involved simulating the needle experience by self-injecting a placebo. All participants (regardless of the needle-experience group) received an Instructions for Use (IFU) leaflet on AI for review prior to self-injection prior to irolizumab administration. Subjects were randomly assigned to administer study drug to their abdomen or anterior thigh area. All participants were self-administered a single SC dose of ilozumab on study day 1 in a simulated home setting ( see Figure 14). Participants were monitored during self-injection, discharged on study day 3, and returned for follow-up visits (study completion) on study days 8, 29, 43, 57, and 85.

Participants Eligible participants are between the ages of 18 and 65, have a Body Mass Index (BMI) between 18.0 - 32.0 kg/ ^m2 (inclusive), and should be in good health with no significant medical history or Abnormal laboratory test. Males and females were recruited with a target of 55% to 60% male participants to simulate the gender distribution of IBD patients. Participants with prior use of any anti-integrin therapy (including irolizumab) or immunosuppressive drugs and with a recent history of corticosteroid use were excluded. Participants with a history of tuberculosis were also excluded.

Procedure . Participants assigned to the needle-experienced group received training (simulated self-injection experience) for 5 and 7 days prior to ibolizumab injection. During the training, the participants were instructed by a healthcare professional in the use of needles and syringes. Following this instruction, participants practiced self-injection 3 times with a placebo solution using a needle and syringe. Participants who experienced the needle (based on their interaction with the syringe) as deemed appropriate by the healthcare professional progressed in the study. Needle-experienced and needle-naive participants were randomized, graded according to gender and needle experience, for injection into the abdomen or anterior thigh area. On Study Day 1, participants self-administered a single SC dose of 105 mg of irolizumab into their abdomen or anterior thigh area using the AI. Participants were assessed for operational difficulties and usage errors and reported pain during and immediately following the injection. A serum sample was obtained 7 days after SC injection (study day 8) for exploratory pharmacokinetic evaluation.

Pain Evaluation Methods . Pain was evaluated via 2 independent methods, both administered by study site personnel. The 7-point categorical Verbal Description Scale (VDS-7) was the primary pain measure used in this study. During administration of VDS-7, patients were asked to select a number from 1 to 7 that best represented pain associated with the injection (on the following scale: 1 = no pain, 2 = very mild pain, 3 = mild pain, 4 = not very painful) severe pain, 5=very severe pain, 6=very severe pain, 7=barely unbearable pain). As a confirmatory assessment, pain was also assessed via a 100-point continuous visual analog scale (VAS). For VAS, patients were asked to mark a line on a horizontal 100 mm scale that best represented their pain (scales as follows: 0 mm = no pain, 100 mm = worst pain). The researchers then measured the distance between the 0 mm point and the patient's marker to determine their VAS score.

Results . The primary endpoint was the proportion of participants with greater than mild pain (VDS-7 > 3) immediately after injection. To achieve the primary endpoint, the upper bound of the 2-sided 95% confidence interval (CI) for the proportion of participants experiencing greater than mild pain immediately after injection must not exceed 30%. Secondary endpoints included the proportion of participants who experienced greater than mild pain at 5, 10, 20, 60 and 240 minutes (4 hours) after injection and the proportion of participants over time for each VDS-7 category. In this study, tolerance was intensively assessed by active monitoring of injection site reactions (ISR) on study day 1 at 5, 60 and 240 minutes after injection and on study days 2, 8, 43 and 85. To identify ISR, a local injection site symptom assessment (LISSA) was performed to assess the magnitude of burning, pruritus, congestion, redness and/or urticaria formation and reaction (if present). All ISRs were classified and reported as adverse events (AEs) or serious AEs as appropriate. Document usage errors and operational difficulties during the use of AI. Additionally, participants' knowledge of IFU and overall opinions on their AI experience were collected. Safety was assessed via AE monitoring, laboratory evaluation, vital signs, physical examination, electrocardiogram (ECG), and immunogenicity. In this study, no formal statistical tests were planned. The variability of the determined PK at a single time point from study day 8 post-injection was assessed as an exploratory endpoint.

result

Thirty healthy participants were recruited and randomized (graded by gender and needle experience) to inject irolizumab into the abdomen or anterior thigh area. All participants completed the study; however, 1 volunteer (who received the needle, thigh injection) did not receive the full dose of ilozumab due to a usage error (discussed separately). The recruited population is broadly representative of the IBD population. The median age of recruited participants was 36 years, mean BMI was 26.1 kg/m ² , and the majority of participants were Caucasian (60%) and not Hispanic or Latino (83%). About half (47%) of the participants were male.

Pain and Tolerance . Half of the participants reported no pain at any time point after injection ( see Figure 15). Of the participants who reported pain, all but one reported "very mild" or "mild" pain, with most pain subsiding within 60 minutes of drug administration. One volunteer reported more than mild pain (VDS-7 = 5) immediately after injection, which subsided to mild pain 5 minutes after injection. Similar data was reported when using VAS (data not shown). Reported pain varied between injection sites. A greater proportion of participants reported pain when injected into the thigh than when injected into the abdomen (60% vs 40%, respectively) ( see Figure 16). One volunteer reporting more than mild pain was assigned to the thigh administration group. Participants who injected into the thigh also reported longer duration of pain than those who injected into the abdomen; all pain experienced after abdominal injection subsided within 5 minutes, and most pain after thigh injection was less than 60 minutes. subsides within minutes. Needle experience training did not appear to affect reported pain following ibolizumab injection. Using the LISSA-based intensive monitoring protocol described above, 40% of participants (12/30) experienced ISR during the study, all within 1 hour of elezolizumab injection (Table 1).

Table 1. Summary of local injection site symptom assessments over time by injection site and needle experience group ISR time point, min Reaction size, mm abdomen thigh Total (n = 30) Unexperienced needles ( n = 6) Experience needles ( n = 9) Total ( n = 15) Unexperienced needles ( n = 6) Experience needles ( n = 9) Total ( n = 15) form hives 60 18 1 (11.1) 1 (6.7) 1 (3.3) redness 5 18 1 (16.7) 1 (6.7) 1 (3.3) twenty one 1 (11.1) 1 (6.7) 2 (33.3) 2 (13.3) 3 (10.0) twenty four 1 (16.7) 1 (6.7) 1 (3.3) 60 <18 1 (11.1) 1 (6.7) 1 (3.3) 18 1 (11.1) 1 (6.7) 1 (3.3) twenty four 2 (23.3) 1 (11.1) 3 (20.0) 3 (10.0) 31 1 (16.7) 1 (6.7) 1 (3.3) ISR injection site reactions

All reported ISRs were mild (grade 1) and transient; all ISRs resolved by study completion. The most frequent ISR is redness, which ranges in diameter from < 18 mm to 31 mm. Most ISR resolves within 60 minutes of injection. One volunteer reported the formation of urticaria (18 mm in diameter) at the abdominal injection site 60 minutes after administration; these urticaria resolved within 3 hours without treatment. Injection site does not appear to affect the frequency or severity of ISR.

Error in use . Twenty-seven of the 30 participants (90%) were able to successfully self-administer ilozumab using the AI without significant error in use, with or without needle experience training. No complaints about AI were recorded, and no patterns of usage errors were observed. A total of 3 usage errors were observed during the study, of which only 1 occurred during the post-injection period. One volunteer initiated AI removal prematurely during the injection, allowing droplets to remain on the volunteer's skin. Of the 2 usage errors that did not occur during injection, 1 volunteer was unsure when to remove the cap from the AI, and the other volunteer incorrectly reported the mock deadline. These 2 usage errors were related to misinterpretation of AI labeling and IFU; neither of these errors affected the dose of ivolizumab administered. Most participants noted that they found AI easy to use. Participants reported that auditory and visual feedback structures were helpful in determining the start and stop times of injections and verifying that the full dose had been administered. However, some participants reported difficulty viewing the visual gyroscope when injected into their abdomen. During the IFU comprehension survey, some confusion was found related to acceptable injection sites, dose preheat time, and product storage.

Pharmacokinetics ( exploratory ). On study day 8 (7 days post-injection), the mean (± SD) serum concentration of ivolizumab in all participants was 13.6 (± 3.66) µg/mL (median is 13.8). Serum concentrations ranged from 5.8 µg/mL to 20.0 µg/mL, with a between-participant variability of approximately 31%. Based on the limited data set, neither injection site nor needle training appeared to affect serum evolizumab concentrations on day 8.

safety. Twenty-nine participants (97%) received the full 105 mg dose of ivolizumab; 1 volunteer received approximately 90% of the 105 mg dose. In conclusion, a single 105 mg SC dose of ivolizumab was safe and well tolerated when self-administered using an AI. About half of the participants experienced treatment-emergent adverse events (TEAEs), mostly related to the injection site. All TEAEs were mild (grade 1) in severity and resolved by study completion. During this study, no significant changes were found in clinical laboratory assessments, vital sign measurements, body weight measurements, or 12-lead ECG. Interestingly, participants injected into the abdomen reported more TEAEs (19 and 9 TEAEs, respectively) than those injected into the thigh. Acupuncture-experienced participants reported less TEAEs than non-acupuncture-experienced participants. A summary of TEAEs can be found in Table 2.

Table 2. Summary of treatment-emergent adverse events abdomen thigh Total ( n = 30) Unexperienced needles ( n = 6) Experience needles ( n = 9) Total ( n = 15) Unexperienced needles ( n = 6) Experience needles ( n = 9) Total ( n = 15) Participants with any TEAE 3 (50.0) 5 (55.6) 8 (53.3) 5 (83.3) 3 (33.3) 8 (53.3) 16 (53.3) Number of TEAEs 13 6 19 6 3 9 28 Suspected to be caused by study drug 2 (33.3) 2 (22.2) 4 (26.7) 3 (50.0) 1 (11.1) 4 (26.7) 8 (26.7) other causes 2 (33.3) 3 (33.3) 5 (33.3) 2 (33.3) 2 (22.2) 4 (26.7) 9 (30.0) severity mild (level 1) 3 (50.0) 5 (55.6) 8 (53.3) 5 (83.3) 3 (33.3) 8 (53.3) 16 (53.3) total 3 (50.0) 5 (55.6) 8 (53.3) 5 (83.3) 3 (33.3) 8 (53.3) 16 (53.3) The value is n (%). Adverse events in TEAE treatment

discuss

In healthy participants, a single, self-administered, SC dose of irolizumab with AI was well tolerated and produced mild pain in the majority of participants. The study met its primary endpoint and only one volunteer experienced more than mild pain after injection. Overall, the data presented here are consistent with AI data for the treatment of other chronic diseases, including rheumatoid arthritis (RA) and chronic kidney disease. These studies show that many patients prefer the convenience of AI over PFS. Patients typically report that AI is less painful than PFS devices and would consider AI to be more portable and easier to use (Kivitz et al, Clin Ther . 2006;28(10):1619–29; Kivitz and Segurado, Expert Rev Med Devices 2007;4(2):109-16; Borras-Blasco et al., Expert Opin Biol Ther . 2010;10(3):301-7; Lim et al., Clin Ther . 2012;34(9):1948- 53). A recent study in UC patients reported similar findings, noting that about three-quarters of the patients in the study preferred AI injections compared to PFS (Vermeire et al. Patient Prefer Adherence . 2018;12:1193-202). In a recent multinational survey, 200 RA patients and 100 nurses were asked to rate the relative importance of each AI component (Tischer and Mehl, Patient Prefer Adherence . 2018;12:1413-24). Ease of use of a pen to perform self-injection (ie, auto-injector) was cited as the most important attribute by both patients and nurses. Other key attributes reported by patients and nurses include "needle safely concealed in syringe body", "auditory feedback after injection is completed", and "visual feedback after injection is completed"; all of these characteristics are attributed to Aerozu Monoclonal antibody AI. Similar results were reported in a European study of 220 RA patients (Thakur et al., Rheumatol Ther . 2016;3(2):245-56). Notably, the proportion of patients in this study who experienced mild ISR was higher than that observed in the Phase 2 EUCALYPTUS study of irolizumab administered via vial and syringe (Vermeire et al., Lancet . 2014;384( 9940):309–18). It is believed that this may reflect differences in study design, which is why investigational intensive assessment of tolerance by actively monitoring ISR at predetermined intervals using LISSA may lead to overreporting of ISR. This first-in-human study is relatively unique in that it combines the assessment of tolerability, assessment of practical human factors (eg, use error), and initial PK assessment into a single trial. This novel approach aims, in part, to assess the overall risk associated with AI in a manner that minimizes the number of clinical studies required, thereby reducing the overall time and cost of AI research and development. PK assessment was incorporated into the study protocol, in which a single blood sample was obtained on study day 8 (approximately at maximum serum concentration following a single SC dose). The purpose of this exploratory PK evaluation was to understand the inter-subject exposure variability following irolizumab delivery by AI SC. In addition, these preliminary PK data help to assess potential differences in exposure after AI injection in UC patients compared to predicted exposure using a model generated based on PK data for administration using vials and syringes. It should be noted that the observed day 8 evolizumab exposure in this analysis was higher than predicted (predicted day 8 median ebolizumab serum concentration ≈7.9 µg/mL [90% CI 4.15–16.3], data not shown) about 75%. The PK variability and unexpectedly higher day 8 exposures in this analysis informed the decision to conduct a 2-part study comparing the pharmacokinetics of ivolizumab delivered by AI and PFS-NSD in healthy volunteers ( See Figure 17 and Example 3). The results of this study effectively eliminate the need for other AI convenience and/or clinical studies. In addition, these results influenced the design of subsequent studies to compare the PK properties between PFS-NSD administration and AI administration, thereby reducing the risk of failure of comparable studies and minimizing exposure to biotherapeutics in healthy volunteers. Unnecessary exposure. Because of the PK findings reported here, a lead panel was added to the original proposed single-part device PK comparability study design. The results of this pilot panel were used to optimize the design of the key panel by informing the appropriate sample size, sample collection duration and body weight range. Information obtained from the human factors portion of this study prompted a small revision of the IFU prior to the PK comparability study. The results of device PK comparability are reported in Example 3.

in conclusion

The results of this study confirmed that a single SC injection of ivolizumab with AI was well tolerated in healthy volunteers, and the pain level after injection was tolerable. Most participants found AI easy to use and experienced only minimal usage errors. AI may be an appropriate delivery structure for certain IBD patients who desire the safety and convenience of self-injection using an invisible needle. The positive results of this initial human tolerability study and the data collected during the subsequent 2-part PK comparability study constitute a complete development program to support the use of the AI in irolizumab-treated patients.

example 3 – Comparable Pharmacokinetics, Safety and Tolerability of Irolizumab Administered via Prefilled Syringe or Auto-Injector in a Randomized Trial in Healthy Volunteers

SUMMARY OF THE INVENTION

Irolizumab is a novel, dual-acting anti-beta7 integrin antibody being studied in several Phase 3 trials in patients with inflammatory bowel disease. An auto-injector (AI) device was also developed to complement the pre-filled syringe (PFS) with needle safety device (NSD) used for subcutaneous administration in these trials. This example demonstrates the comparability of the pharmacokinetics (PK), tolerability and safety of the two devices.

This randomized, open-label, 2-part study in healthy participants assessed the comparability of ebolizumab exposure between AI and PFS-NSD. Part 1 (pilot) involved a small number of participants and used initial results to complete the study design of the larger Part 2 (key). In both parts, participants were randomly assigned to receive a single dose of 105 mg subcutaneous (SC) irolizumab via AI or PFS-NSD. Randomization was graded according to body weight. The primary PK results were _Cmax , _AUClast and AUC0 _-inf .

180 healthy participants (Part 1: n = 30, Part 2: n = 150) received a single SC dose of ilozumab via AI or PFS-NSD. The primary PK results from Part 1 support a modification to the Part 2 study design. Part 2 results confirmed that the exposure of ivolizumab between devices was equivalent, with a geometric mean ratio (GMR) of _Cmax between AI and PFS-NSD of 102% and a geometric mean ratio of _AUClast of 102% 98.0%, and the geometric mean ratio of AUC _0-inf was 97.6%. Median _Tmax and mean terminal t1 _/2 were also similar between devices. GMRs and 90% confidence intervals for all major PK parameters were well within predetermined equivalence limits (80% to 125%).

This PK study confirmed that a single SC injection of 105 mg of elezolizumab with AI or PFS-NSD resulted in equivalent elocizumab exposure and similar safety and tolerability in healthy participants. Taken together, these results support the use of AI for the administration of irolizumab.

Introduction

The PK comparability study presented in this example (NCT02996019) was designed to demonstrate the comparability of elezolizumab exposure following administration with AI and PFS-NSD, and to evaluate elezolizumab following SC injection using both devices safety and tolerability. Part 1 of the study was an exploratory pilot group to assess the geometric mean ratio (GMR) and variability of PK parameters when administered with AI versus PFS-NSD. These results informed the study design for Part 2 (Key Group), including sample size and study duration. In Part 2, the study was designed to demonstrate comparability of exposure between single doses of irolizumab administered via AI or PFS-NSD. The PK comparability study presented in this example utilizes the exploratory PK results of the tolerability study presented in Example 2 to improve the study design and final protocol.

method

Study Design and Procedures . This study was a randomized, multicenter, open-label, parallel group study conducted in healthy participants at three clinical sites in the United States ( see Figure 18). This 2-part study consisted of a lead panel (Part 1) and a pivot panel (Part 2), where the sample size was sufficient to detect, with 80% power, the difference in exposure, if any, between the 2 device groups. In both parts, healthy participants were randomly assigned 1:1 to receive a single dose of 105 mg SC ebolizumab via AI (test device) or PFS-NSD (reference device). Irolizumab was administered to the participant's abdomen by a healthcare professional (HCP). Randomization in both groups was graded according to body weight (≤ 79.9 vs ≥ 80 kg).

Participants Eligible healthy participants included males and females between the ages of 18 and 55 with a body mass index (BMI) between 18.0 - 30.0 kg/m ² . Based on the results of the pilot group, participants in the pivotal group were also required to have a body weight in the range of 60 kg to 100 kg (inclusive) at study entry. Participants must be in good health (no clinically significant findings on medical history, physical 12-lead ECG, or vital signs). Participants who had been previously exposed to any immunosuppressive or anti-integrin therapy, including irolizumab, were excluded.

Evaluation and Outcome Measures . Blood samples were collected before dosing and 6 hours after dosing on day 1, then on days 2, 4, 6, 8, 11, 15, 29, 43, 57, and 71 (study completion). Irolizumab serum concentrations were determined in Parts 1 and 2. Geometric mean ratios (GMRs) and variability of ilotezumab were measured in Part 1: maximum ebolizumab concentration ( _Cmax ), concentration from time of drug administration to last measurable concentration - Area under the time curve (AUC _last ), AUC extrapolated to infinity (AUC _0-inf ) and the ratio of AUC _last to AUC _0-inf (AUCR). In part 2, _Cmax , _AUClast and AUC0 _-inf were measured as primary endpoints. Secondary PK parameters included time to maximum ivolizumab concentration ( _tmax ), terminal elimination half-life (t1 _/2 ), and AUCR. Before dosing on day 1 and on days 29, 57 and 71, blood samples were collected for anti-drug antibody (ADA) determination in Part 1 and Part 2. Data from all ADA-positive participants were included in the final PK statistical analysis unless participants met the predetermined exclusion criteria for PK analysis. Safety and tolerability assessments included the incidence, nature, and severity of adverse events, graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Incidence of injection site reactions, changes in vital signs, physical examination findings, clinical laboratory results, and incidence of ADA were also evaluated.

Bioanalytical Methods . Irolizumab concentrations were measured using a validated immunoassay (ICON). This Gyrolab fluoroimmunoassay utilizes a minimum required dilution (MRD) of 1/100, and the minimum quantifiable irolizumab concentration in UC, CD, and healthy volunteer sera is 80 ng/mL. A validation assay was used to detect anti-ivolizumab antibodies in serum. This colorimetric ELISA uses 1/20 MRD and a monoclonal anti-ivolizumab control antibody. The relative sensitivity of this method in healthy volunteer serum was determined to be 12.0 ng/mL. To establish drug tolerance for the assay: 28 ng/mL of positive control ADA was detected in the presence of 50 μg/mL of irolizumab.

Sample size determination . Up to 30 healthy participants are planned to be recruited in Part 1 to ensure that at least 12 participants in each group (24 in total) have an evaluable PK profile, enabling estimation of PK parameters ( _Cmax ) , AUC _last and AUC _0-inf ) GMR and coefficient of variation (CV%). Part 2 plans to recruit 146 participants. Assuming a dropout rate or non-evaluable PK profile of approximately 10% of participants by Day 71, a total of approximately 131 healthy participants with an evaluable intact PK profile expected to have at least 80% efficacy based on the Part 1 pilot panel The GMR and PK variability results were used to demonstrate exposure comparability of _Cmax , _AUClast and AUC0 _-inf .

Pharmacokinetic Analysis . PK parameters were determined from serum irolizumab concentrations using a non-compartmental approach (NCA) and performed using Phoenix WinNolin (Centara USA, Inc., version 6.4) .

Statistical Analysis . The analysis population consisted of all participants who received SC ibolizumab injections and had an evaluable PK profile, defined as a sample with sufficient quantities for accurate determination of key PK parameters. Specifically, participants who terminated early on or before Day 15 were deemed not to have an evaluable PK profile. Participants with no samples available for determination of concentrations on day 71 were excluded from statistical analysis of AUC _last ; participants with <3 samples available on days 28, 43, 51 and 71 were excluded from statistical analysis of AUC _0-inf Scientific analysis excluded. At the discretion of the study and commissioned clinical pharmacokinetic/biostatistician, participants with pre-dose concentrations >5% _Cmax may be excluded from statistical analysis of all PK parameters. Descriptive, exploratory analyses of PK parameters were performed using data from Part 1 (lead), with emphasis on assessing GMR, CV%, and AUCR distributions to inform Part 2 (key) sample size and final study design. Only data from the key group (Part 2) were included in the comparable statistical analysis. In Part 2, an analysis of variance (including treatment as a fixed effect) was performed to evaluate the comparability of _Cmax , _AUClast and AUC0 _-inf between the AI group and the PFS-NSD group. Data for _Cmax , _AUClast , and AUC0 _-inf were natural log (ln)-transformed prior to analysis, and 90% confidence intervals (CIs) for GMR in the AI group relative to the PFS-NSD group were obtained by taking the mean The inverse log (log scale) of the corresponding 90% CI of the difference was calculated. Exposures between the AI and PFS-NSD groups met the PK comparability criterion if the 90% CIs for GMR for _Cmax , _AUClast , and AUC0 _-inf were all within 80% to 125%.

result

Pharmacokinetics - Pilot Study ( Part 1 ). All 30 participants recruited and randomized into Part 1 received ilozumab via AI (n = 15) or PFS-NSD (n = 15) A single 105 mg SC dose of the antibody. 27 participants completed the study; 2 participants (1 in each group) discontinued early due to loss of follow-up and 1 discontinued due to serious AE (flare) 32 days after ivolizumab (PFS- NSD group); this AE was not considered to be related to ivolizumab treatment. Participant characteristics are shown in Table 3.

Table 3. Participant Characteristics Pilot Group ( Part 1 ) Key Groups ( Part 2 ) AI n = 15 PFS-NSD n = 15 AI n = 74 PFS-NSD n = 76 Average age (min, max), years 42 (28, 54) 34 (20, 53) 35 (21, 55) 37 (19, 55) Average body weight (min, max), kg 79.7 (65.1, 108.8) 74.3 (50.8, 92.1) 76.2 (61.0, 99.4) 76.3 (55.90, 97.5) Average height (min, max), cm 172.0 (154.3, 190.8) 173.5 (154.6, 194.9) 170.3 (151.2, 193.4) 170.7 (151.5, 189.0) Average BMI (min, max), kg/m ² 26.8 (23.2, 29.9) 24.5 (20.6, 29.2) 26.3 (20.4, 30.0) 26.2 (19.2, 29.9) Gender, n (%) male 9 (60.0) 10 (66.7) 40 (54.1) 40 (52.6) female 6 (40.0) 5 (33.3) 34 (45.9) 36 (47.4) Race, n (%) Asian 0 0 2 (2.7) 1 (1.3) Black/African American 5 (33.3) 4 (26.7) 31 (41.9) 23 (30.3) white people 10 (66.7) 11 (73.3) 40 (54.1) 51 (67.1) unknown 0 0 1 (1.4) 1 (1.3) Nationality, n (%) Hispanic or Latino 6 (40.0) 3 (20.0) 14 (18.9) 14 (18.4) non-Hispanic or Latino 9 (60.0) 12 (80.0) 60 (81.1) 62 (81.6) JCV antibody – positive, n (%) 7 (46.7) 8 (53.3) 38 (51.4) 46 (60.5) AI : Auto-Injector. BMI: Body Mass Index. JCV : John Cunningham virus. PFS-NSD : Prefilled syringe with needle safety device.

In the entire group, the majority of participants were male (63.3%) and Caucasian (70.0%). The weight and age of the participants were not well balanced across treatment groups; the mean weight in the AI group was 79.7 kg and the mean weight in the PFS-NSD group was 74.3 kg, and the mean age in the AI group was 42 years and The mean age in the PFS-NSD group was 34 years. Since body weight appears to affect ivolizumab exposure, especially AUC _0-inf ( see Figure 19), body weight imbalance between treatment groups could potentially bias the assessment of GMR values and PK variability for key PK parameters . Therefore, participants in the lead group who weighed less than 60 kg (n=4) or more than 100 kg (n=1) were excluded from the exploratory analysis, which was designed to assess use to guide the identification of pivotal studies (p. Part 2) GMR and CV% of PK parameters for final sample size. The PK-evaluable populations meeting the weight limit of 60 kg to 100 kg in Part 1 included 14 participants in the AI group and 11 participants in the PFS-NSD group. As expected, when balancing the body weight range between the two arms by restricting body weight to the range of 60-100 kg, GMR values for the main PK parameters _Cmax , _AUClast and AUC0 _-inf in the AI and PFS-NSD groups, respectively were 0.95, 1.02 and 1.02 (Table 4).

Table 4. Summary of PK parameters in Group 1 participants meeting the 60 kg to 100 kg weight limit parameter AI (test) PFS-NSD (reference) GMR (%) Aggregated CV% n ^a geometric mean ^b n ^a geometric mean ^b _Cmax (µg/mL) 14 10.6 11 11.1 0.955 30.1 AUC _last (day∙µg/mL) 12 273.3 9 267.9 1.020 34.1 AUC _0-inf (day∙µg/mL) 14 284.6 10 278 1.024 34.0 AI : Auto-Injector. ANOVA : Analysis of variance. AUC _0-inf : AUC extrapolated to infinity. _AUClast : Area under the concentration-time curve from the time of drug administration to the last measurable concentration ( _tlast for all available data is day 71). _Cmax : Maximum concentration. GMR: Geometric Mean Ratio. PFS-NSD : Prefilled syringe with needle safety device. ^a : Number of observations per treatment eligible for analysis. ^b : The geometric mean is based on the least squares mean of the _Cmax and AUC parameters in ANOVA calculated by converting the natural log mean back to a linear scale.

Therefore, the sample size of the key panel was calculated based on these GMR and CV% values, and a weight restriction was added to the Part 2 study as an inclusion criterion. In addition, all participants had AUCR values > 80% (data not shown), which met the US Food and Drug Administration (FDA) guidelines (US Department of Health and Human Services. Guidance for industry: statistical approaches to establishing bioequivalence. The need for bioequivalence studies as stated in 4 March 2020), and thereby prompted the change in the final date of the pivotal study from day 85 to day 71 as originally planned.

Pharmacokinetics - Pivotal Study ( Part 2 ) . In pivotal group, 150 (100%) recruited and randomized participants received Airlot via AI (n = 74) or PFS-NSD (n = 76) A single SC 105 mg dose of fuzumab. 8 participants discontinued the study; 5 due to loss to follow-up (3 in the AI group and 2 in the PFS-NSD group), 1 due to protocol violation for not meeting weight guidelines (PFS-NSD), and 2 (1 per group) due to participant withdrawal. Throughout Part 2 (including both groups), 53.5% of the participants were male, 60.7% were Caucasian, and 36.0% were African American. Well balanced body weight and age in treatment groups; mean body weight in AI group was 76.2 kg and mean body weight in PFS-NSD group was 76.3 kg, and mean age in AI group was 35 years and mean age in PFS-NSD group was 37 years old (Table 3).

Changes in serum concentrations of evolizumab over time for the AI and PFS-NSD groups are shown in Figure 20; PK parameters are summarized in Table 5. GMR (90% CI) for C _max between AI and PFS-NSD groups was 102% (94.2–111%), AUC _last was 98.0% (89.3–107%), and AUC _0-inf was 97.6% ( 88.6–107%) (Table 6).

Table 5. Summary of PK parameters for ivolizumab in pilot and pivotal studies parameter Pilot Group ( Part 1 ) ^a Key Groups ( Part 2 ) n AI (n = 14) n PFS-NSD (n = 11) n AI (n = 76) n PFS-NSD (n = 74) _Cmax (µg/mL) 14 10.6 (28.3%) 10 11.0 (35.3%) 73 12.1 (32.0%) 73 12.2 (28.3%) t _max ^b (days) 14 5.00 (2.95, 9.94) 10 3.98 (2.90, 6.97) 73 5.04 (2.98, 14.0) 73 6.97 (3.00, 14.0) AUC _last (day∙µg/mL) 13 272 (39.8%) 9 268 (27.0%) 69 319 (35.3%) 72 325 (33.1%) AUC _0-inf (day∙µg/mL) 13 284 (41.6%) 10 278 (27.0%) 69 329 (36.0%) 72 337 (35.2%) t _1/2 ^c (day) 13 12.2 (4.90) 10 11.4 (4.04) 69 11.8 (3.85) 72 12.2 (4.39) CL/F (L/day) 13 0.369 (41.6%) 10 0.378 (27.0%) 69 0.319 (36.0%) 72 0.311 (35.2%) ^AUCRd 13 0.957 (3.5%) 9 0.975 (2.6%) 67 0.966 (3.4%) 72 0.964 (3.7%) ≤80% 0 0 0 1 (1%) >80% 13 (100%) 9 (100%) 67 (100%) 71 (99%) AI : Auto-Injector. AUC _0-inf : AUC extrapolated to infinity. _AUClast : The area under the concentration-time curve from the time of drug administration to the last measurable concentration. AUCR : The ratio of AUC _last to AUC _0-inf . _Cmax : Maximum concentration. PFS-NSD : Prefilled syringe with needle safety device. PK : Pharmacokinetics. t _1/2 : terminal elimination half-life. _tmax : Time to maximum concentration. Geometric mean (geometric CV%) data are presented unless otherwise indicated. ^aOnly participants who met the weight limit (60 kg to 100 kg) were included in the pilot group. ^b presents the median value of t _max (min, max). ^c presents the arithmetic mean (SD) of t _1/2 . ^d presents n (%).

Table 6. Results of ANOVA to evaluate comparability of PK parameters of AI and PFS-NSD (key panel) parameter AI ( test) PFS-NSD ( reference) GMR ^c (%) GMR 90% CI ^d (%) n ^a geometric mean ^b n ^a geometric mean ^b _Cmax (µg/mL) 73 12.5 73 12.2 102 94.2-111 AUC _last (day∙µg/mL) 69 319 72 325 98.0 89.3-107 AUC _0-inf (day∙µg/mL) 69 329 72 337 97.6 88.6-107 AI : Auto-Injector. ANOVA : Analysis of variance. AUC _0-inf : AUC extrapolated to infinity. _AUClast : Area under the concentration-time curve from the time of drug administration to the last measurable concentration. _Cmax : Maximum concentration. GMR: Geometric Mean Ratio. PFS-NSD : Prefilled syringe with needle safety device. ^a : Number of observations per treatment eligible for analysis. ^b : The geometric mean is based on the least squares mean of the _Cmax and AUC parameters in ANOVA calculated by converting the natural log mean back to a linear scale. ^c The geometric mean ratio (expressed as a percentage) of the test/reference ratio of the parameter mean of the natural log conversion parameter. Convert the natural log conversion ratio back to a linear scale. ^d 90% confidence interval (expressed as a percentage) of the ratio of the parameter means for the natural log conversion parameter. Converts the natural logarithm scaling confidence limits back to a linear scale.

The 90% CI of the GMR for each of these key PK parameters was within the predetermined equivalence limits of 80% to 125%, which met the predetermined comparability criteria and supported air equivalence between the 2 device groups benzumab exposure. The results also confirmed that the AI and PFS-NSD groups had similar median time to maximum observed concentration (t _max , 5.04 days vs 6.97 days) and mean terminal elimination half-life (t _1/2 , 11.8 days vs 12.2 days) ).

safety. The overall incidence of treatment-emergent ADA was 20.7% (6/29) in the post-baseline evaluable participants in the lead group and 29.7% (44/148) in the key group, and in the lead group (3/148) 15 [20%] vs 3/14 [21.4%]) and the pivotal group (20/73 [27.4%] vs 24/75 [32.0%]) when comparing the AI and PFS-NSD groups with similar rates. The PK profile of ADA+ subjects appeared to be similar to their ADA negative subjects ( see Figure 21). Treatment-emergent adverse events (TEAEs) were experienced by 53% of participants in the lead group and 35% in the pivotal group, mostly of mild severity (Table 7).

Table 7. Treatment-emergent adverse events (TEAEs) Pilot Group ( Part 1 ) Key Groups ( Part 2 ) Total ( Parts 1 and 2 ) n = 180 AI n = 15 PFS-NSD n = 15 Total n = 30 AI n = 74 PFS-NSD n = 76 Total n = 150 any TEAE 9 (60.0) 7 (46.7) 16 (53.3) 22 (29.7) 27 (35.5) 49 (32.7) 65 (36.1) Number of TEAEs twenty two 7 29 32 45 77 106 any SAE 0 1 (6.7) 1 (3.3) 0 0 0 1 (0.6) Any ISR related TEAE 5 (33.3) 4 (26.7) 9 (30.0) 1 (1.4) 0 1 (0.7) 10 (5.6) suspected by caused by study drug 6 (40.0) 3 (20.0) 9 (30.0) 7 (9.5) 10 (13.2) 17 (11.3) 26 (14.4) other causes 7 (46.7) 2 (26.7) 11 (36.7) 18 (24.3) 21 (27.6) 39 (26.0) 50 (27.8) Concomitant conditions 4 (26.7) 0 4 (13.3) 7 (9.5) 14 (18.4) 21 (14.0) 25 (13.9) accompanying drug 0 0 0 2 (2.7) 1 (1.3) 3 (2.0) 3 (1.7) program 3 (20.0) 1 (6.7) 4 (13.3) 0 1 (1.3) 1 (0.7) 5 (2.8) other 2 (13.3) 3 (20.0) 5 (16.7) 10 (13.5) 6 (7.9) 16 (10.7) 21 (11.7) missing 0 0 0 1 (1.4) 0 1 (0.7) 1 (0.6) Most Severe NCI CTCAE Grade for AE Level 1 8 (53.3) 4 (26.7) 12 (40.0) 19 (25.7) 27 (35.5) 46 (30.7) 58 (32.2) Level 2 1 (6.7) 2 (13.3) 3 (10.0) 3 (4.1) 0 3 (2.0) 6 (3.3) Level 3 0 1 (6.7) 1 (3.3) 0 0 0 1 (0.6) total 9 (60.0) 7 (46.7) 16 (53.3) 22 (29.7) 27 (35.5) 49 (32.7) 65 (36.1) AE : Adverse Events; AI : Auto-Injector, ISR : Injection Site Reactions, NCI CTCAE : National Cancer Institute Common Terminology Criteria for Adverse Events, PFS-NSD : Prefilled Syringe with Needle Safety Device, SAE : Serious Adverse Events, TEAE : Treatment-emergent adverse events, unless otherwise specified, data are reported as n (%).

One participant in the PFS-NSD arm of the lead group had a serious AE (grade 3 onset) approximately 32 days post-injection that led to study discontinuation and was not considered related to ibolizumab treatment. Nine participants in the lead group (5 with AI and 4 with PFS-NSD) and 1 in the pivot group (AI group) had injection site reactions reported as TEAEs. Six (40.0%) participants in the AI group and 3 (20.0%) in the PFS-NSD group experienced treatment-related TEAEs in the lead group and 7 (9.5%) in the AI group in the key group ) participants and 10 (13.2%) participants in the PFS-NSD group.

discuss

The pivotal panel in this study demonstrated comparable elrolizumab exposures between the AI and PFS-NSD groups following a single dose of SC elezolizumab in healthy participants. The observed GMRs for each primary PK parameter were between 98% and 102% with 90% CIs within predetermined equivalence limits. These GMRs for all major exposure parameters were surprising considering the GMRs obtained when comparing other AIs and PFS-NSDs in healthy participants. For example, a recent study comparing AI and PFS devices for SC administration of an adalimumab biosimilar (SB5) showed GMRs of 102%, 107% for _Cmax , _AUClast and _AUCinf , respectively and 110% with a 90% CI within the equivalence limits of 80% to 125%. In a similar study of the adalimumab biosimilar BI 695501, the AUC _0-inf was 100% (90% CI 82.1–122.3%) and the _Cmax was 110% (90% CI 96.8–125.4%), which was higher than 90% CI upper limit of equivalence of 125% (Voltaire®-AI study) ( ^Ramael et al., Rheumatol Ther . 2018;5(2):403–21; Shin et al., Drug Des Devel Ther . 2018;12: 3799–805). The pilot group was used to inform the final design of the key group. The pilot group primarily assessed GMR and PK parameter variability, which are key assumptions used for sample size estimation. To minimise the risk of underpowered key PK comparability studies, a smaller lead group was added to the original study protocol to determine GMR values for key PK parameters following ivolizumab administration by AI or PFS-NSD and PK variability. The GMR and PK variability values obtained from the lead panel (see Table 4) add confidence in estimating the sample size of the key panel. Despite weight stratification at randomization, the distribution of final body weights in the lead group was uneven, which could bias the final GMR results. To minimize this bias, GMR and PK variability were estimated using only data from participants in the body weight range of 60 kg to 100 kg (a common body weight range for both groups within the lead group). This weight limit of 60 kg to 100 kg was also implemented in key groups as inclusion criteria. The pilot panel also assessed a 10-week (70-day) study duration, which was shorter than the 2-week study duration recommended by the FDA for the original study plan. As expected, all evaluable participants in the lead group had AUCR values >80%, which is required by the FDA in PK comparability studies. This result suggests that the 10-week study duration is long enough to capture more than 80% of AUC _inf and thus the duration of pivotal studies can be shortened from 12 weeks to 10 weeks without an extrapolation of < 20% for calculating AUC _inf Risk of AUC requirements. The immunogenicity in this study was relatively higher than in other studies with ivolizumab. ADA rates were >20% in both the lead and pivotal groups, and 5% (2/38) of UC patients in the phase 1 trial who received single or multiple doses of ilrolizumab (Rutgeerts et al, Gut . 2013; 62(8):1122–30) and 5% (4/81) of patients with UC who received multiple doses of ivolizumab in a randomized phase 2 trial (Vermeire et al, Lancet . 2014;384(9940): 309–18) had detectable ADA following ivolizumab treatment. The incidence of immunogenicity was equal in the PFS-NSD and AI groups in the pilot and pivotal groups. The higher ADA rates in this study may be due to differences in study design: this study used a single SC dose regimen and a study population of healthy participants not taking immunosuppressive drugs. In contrast, patients in previous trials of ivolizumab had poorly controlled moderate to severe UC and were often treated with immunosuppressive agents. Although the incidence of ADA was relatively high (>20%) following single SC injection of ilozumab in this study, considering the PK profile of ADA-positive participants was similar to that observed in ADA-negative participants Most of these overlap, so the effect of ADA positivity on PK appears to be minimal (Figure 21). In addition, in the Part 2 pivotal study, the variability (CV%) in AUC0 _-inf or _AUClast values ranged from 33% to 36%, which was consistent with what they observed for other monoclonal antibodies (Ramael et al. et al., Rheumatol Ther . 2018;5(2):403-21; Anumolu et al., Clin Pharmacol Drug Dev. 2018;7(8):829-36). This small exposure variability further confirms that the total exposure (AUC0 _-inf ) between ADA-positive and ADA-negative participants was very similar. Data from all evaluable participants (regardless of their ADA status) were included in the statistical evaluation of comparability, and the final results demonstrated a high degree of exposure similarity between groups. Bioequivalence testing of AI arm and PFS-NSD arm and sample size determination in pivotal study design after a single SC dose of ivolizumab was based on subgroups of participants from 60 kg to 100 kg body weight data of. Although the finding of comparable ibrolizumab exposure between AI and PFS-NSD was based on data from participants within the defined weight range, it was considered that this study did not identify differences in exposure due to the drug delivery device alone , so it can be applied to individuals outside this weight range. Additionally, a similar study of another therapeutic antibody, which was not body weight limited, suggested that this may be a reasonable assumption (Ramael et al., Rheumatol Ther . 2018;5(2):403-21). In this study, a similar relationship was observed between drug exposure and body weight, but bioequivalence was still achieved when comparing AI and PFS-NSD devices for SC administration of adalimumab, regardless of body weight. Irolizumab is known to have a significant effect on receptor weight clearance (Tang et al., Aliment Pharmacol Ther . 2018;47(11):1440-52), a finding that was confirmed in the lead group of this study. In these healthy participants, a single SC dose of ilozumab was generally safe and well tolerated when administered using AI or PFS-NSD. In the key group, AI and PFS-NSD were comparable in treatment-related TEAEs (9.5% vs 13.2%). Most TEAEs were of mild or moderate severity and most resolved by the end of the study. One serious AE (flare) led to study discontinuation in the PFS-NSD arm of the lead group; however, this AE was not considered to be related to ivolizumab treatment.

in conclusion

The results of this PK comparability study confirmed that evolizumab exposure was similar when administered to SC via AI or PFS-NSD, with 90% CIs for exposure parameters (AUC _last , C _max , AUC _inf ) contained within Within the bioequivalence limit (80–125%) between devices. In addition, a single SC dose of irolizumab administered via AI was generally well tolerated in healthy participants without associated increased adverse events compared to PFS-NSD injection. This study also highlights the value of a pilot group in facilitating the design of pivotal PK comparability studies, as data from the pilot group increases confidence in the study design and minimizes assumption bias.

example 4

This example provides details related to PFS-NSD injection force.

There are multiple force definitions during a user's injection experience. The user does the following: 1) removes the needle cover, and 2) depresses the plunger until the needle safety is activated. Figure 22 shows the different force definitions. Table 8 shows the forces and their limits. Table 9 shows that the injection force is within the limits of the 1mL and 2.25mL configurations.

Table 8 force Limit Average injection force (also known as slip force) ≤26 Newton (N) break free ≤33N maximum injection force ≤33N

Table 9 Attributes Real-time aging (2-8°C) times are shown below in months High temperature (25℃) T=initial T=6 T=12, 24 T=3 T=6 slip force within limits within limits break free within limits within limits maximum injection force within limits within limits

There is a spring mechanism that pushes the liquid out of the auto-injector. Therefore, injection time is a relevant property. In certain embodiments, the injection time is between about 0.4 seconds and about 9 seconds. In certain embodiments, the injection time is about 5 seconds.

Auto-injectors used in the formulations disclosed herein provide favorable injection times and extended stability.

Embodiments of the subject matter disclosed herein

It will be apparent from the foregoing description that changes and modifications of the subject matter disclosed herein can be made to adapt it to various applications and conditions. Such embodiments are also within the scope of the following claims.

The listing of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of embodiments herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

FIG. 1 depicts solubility curves of lauric, myristic, and palmitic acids as a function of pH and polysorbate 20 (PS20) concentration, as described in Example 1. FIG. Figure 2 depicts the effect of arginine on solution viscosity, as described in Example 1. 3 depicts the effect of protein concentration on the viscosity of formulations containing 200 mM arginine succinate, as described in Example 1. FIG. FIG. 4 depicts the change in the percentage of high molecular weight species (HMWS) of ilrolizumab in prefilled syringes with different amounts of silicone oil, as described in Example 1. FIG. The term "high molecular weight species (HMWS)" and the term "high molecular weight form (HMWF)" are used interchangeably herein. FIG. 5 depicts the percent change in ilrolizumab HMWS over time at different tungsten concentrations, as described in Example 1. FIG. Figure 6 depicts the effect of zinc on the viscosity at 25°C of formulations containing 20 mM histidine, 200 mM arginine succinate (pH 5.8) at 25°C, as in Example 1 stated. FIG. 7 shows protein aggregate formation by 50 mM zinc and 150 mg/mL ivolizumab at room temperature, as described in Example 1. FIG. FIG. 8 depicts HMWS formation by 10 mM zinc and 10 mg/mL or 50 mg/mL ivolizumab at 40°C, as described in Example 1. FIG. 9 depicts the effect of histidine on HMWS formation at 40°C for formulations containing 10 mM zinc and 10 mg/mL ivolizumab, as described in Example 1 . 10 depicts the effect of succinate on HMWS formation at 40°C for formulations containing 10 mM zinc and 10 mg/mL elezolizumab, as described in Example 1. FIG. Figure 11 shows different concentrations of histidine and succinate and their combined effect on HMWS formation at 40°C for formulations containing 10 mM zinc and 50 mg/mL ivolizumab, as described in Example 1. FIG. 12 shows example prefilled syringes (top two) and auto-injector (last one), as set forth in Example 2. FIG. Figure 13 shows a prefilled auto-injector of elezolizumab, as described in Example 2. Figure 14 shows the auto-injector (AI) tolerability and human factors study design, as set forth in Example 2. 15 shows a graph indicating pain intensity (7-point visual interpretation scale) as a function of time, as set forth in Example 2. FIG. 16 depicts a graph indicating pain (7-point Visual Elaboration Scale) as a function of time for a certain injection site, as described in Example 2. FIG. Figure 17 shows a two-part pharmacokinetic link study design, as described in Example 2. *Setting the Geometric Mean Ratio (GMR) 1.15 as the decision point is based on the assumption that the 15% difference between prefilled syringes with needle safety devices (PFS-NSD) is likely to be real and thus this study cannot confirm biological Equivalence. ^† N adjustment due to GMR from the pilot study. AI, auto-injector; AUC, area under the curve; _Cmax , maximum serum concentration of ivolizumab. Figure 18 shows participant profiles, as set forth in Example 3. AI , auto-injector; AUC , area under the curve; PFS-NSD , prefilled syringe with needle safety device; PK , pharmacokinetics; SC , subcutaneous. ^aExcluded due to eligibility criteria (weight restriction). Participants were excluded from the specific PK analysis due to insufficient PK data for the calculation. 19 shows the effect of body weight on irolizumab _Cmax (upper panel) or AUCo _-inf (lower panel), as described in Example 3. FIG. AI , auto-injector; AUC , area under the curve; AUC0 _-inf , AUC extrapolated to infinity; _Cmax , maximum concentration; PFS-NSD , prefilled syringe with needle safety; SC , subcutaneous. 20 shows the serum concentrations of ebolizumab as a function of time with AI and PFS-NSD based on a linear scale (A) and a semi-log scale (B) in the pivotal study, as described in Example 3. FIG. AI , automatic injector; PFS-NSD , prefilled syringe with needle safety device. Figure 21 shows the use of AI (N=73 (total), of which 20 subjects were ADA positive, top panel) and PFS-NSD (N=75, of which 24 subjects were ADA positive, bottom panel). Evolizumab serum concentrations as a function of time according to ADA status, as set forth in Example 3. ADA , anti-drug antibody; AI , auto-injector; PFS-NSD , pre-filled syringe with needle safety device. FIG. 22 shows different force definitions during injection, as set forth in Example 4. FIG.

         <![CDATA[<110> Jiannandek Corporation]]> Swiss Merchant F.Hoffmann-La Roche AG Hoffmann-La Roche Inc.<![CDATA[<120> Anti-rectification Catenin β7 Antibody Formulations and Devices]]> <![CDATA[<130> 00B206.1132]]> <![CDATA[<150> US 63/059,427]]> <![CDATA[<151> 2020- 07-31]]> <![CDATA[<160> 12 ]]> <![CDATA[<170> PatentIn v3.5]]> <![CDATA[<210> 1]]> <![CDATA[ <211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > Synthetic Peptides]]> <![CDATA[<400> 1]]> Arg Ala Ser Glu Ser Val Asp Asp Leu Leu His 1 5 10 <![CDATA[<210> 2]]> <![CDATA[< 211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 2]]> Lys Tyr Ala Ser Gln Ser Ile Ser 1 5 <![CDATA[<210> 3]]> <![CDATA[<211> 9]] > <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 3]]> Gln Gln Gly Asn Ser Leu Pro Asn Thr 1 5 <![CDATA[<210> 4]]> <![CDATA[<211> 10]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA [<400> 4]]> Gly Phe Phe Ile Thr Asn Asn Tyr Trp Gly 1 5 10 <![CDATA[<210> 5]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 5]]> Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 1 5 10 15 Ser <![CDATA[<210> 6]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 6]]> Arg Thr Gly Ser Ser Gly Tyr Phe Asp Phe 1 5 10 <![CDATA[<210> 7]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <! [CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 7]]> Ala Arg Thr Gly Ser Ser Gly Tyr Phe Asp Phe 1 5 10 <![CDATA[<210> 8]]> <![CDATA[<211> 108]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 8]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Leu 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Asn 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <![CDATA[<210> 9]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> < ![CDATA[<400> 9]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe Ile Thr Asn Asn 20 25 30 Tyr Trp Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Gly Ser Ser Gly Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 10]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213 > Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400> 10]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Leu 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Asn 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 11]]> <![CDATA[<211> 446]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> <![CDATA[<400 > 11]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe Ile Thr Asn Asn 20 25 30 Tyr Trp Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Gly Ser Ser Gly Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <![CDATA[<210> 12]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic Peptide]]> < ![CDATA[<400> 12]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe Ile Thr Asn Asn 20 25 30 Tyr Trp Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Gly Ser Ser Gly Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
      

Claims (86)

A formulation comprising a monoclonal anti-integrin β7 antibody, wherein the concentration of the anti-integrin β7 antibody is at least about 100 mg/ml, and the formulation has a viscosity at 25°C of less than about 20 centipoise (cP), and wherein the formulation has prolonged stability. The formulation of claim 1, wherein the anti-integrin β7 antibody is a humanized antibody. The formulation of claim 1, wherein the formulation has a viscosity of about 7 cP at 25°C. The formulation of any one of claims 1 to 3, wherein the anti-integrin beta7 antibody comprises three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2, and HVR-L3, and three Heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) The HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. The formulation of any one of claims 1 to 4, wherein the anti-integrin β7 antibody comprises: a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain can be A variable region comprising the amino acid sequence shown in SEQ ID NO:9. The formulation of any one of claims 1 to 5, wherein the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 10 The amino acid sequence shown in ID NO: 11. The formulation of any one of claims 1 to 5, wherein the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 10 The amino acid sequence shown in ID NO: 12. The formulation of any one of claims 1 to 7, wherein the anti-integrin β7 antibody is etrolizumab. The formulation of any one of claims 1 to 8, wherein the concentration of the anti-integrin beta7 antibody in the formulation is between about 100 mg/ml and about 220 mg/ml. The formulation of any one of claims 1 to 9, wherein the concentration of the anti-integrin β7 antibody in the formulation is at or about 150 mg/mL. A formulation as claimed in any one of claims 1 to 11, wherein the pH of the formulation is greater than 5.5. The formulation of claim 12, wherein the pH of the formulation is between 5.65 and 6.1. A formulation as claimed in any one of claims 1 to 12, wherein the pH of the formulation is 5.8, between 5.7 and 5.9 or between 5.75 and 5.85. The formulation of any one of claims 1 to 13, further comprising a surfactant, wherein the concentration of the surfactant in the formulation is between 0.03% w/v and 0.06% w/v. The formulation of claim 14, wherein the concentration of the surfactant in the formulation is at or about 0.04% w/v. The formulation of claim 14 or 15, wherein the surfactant is polysorbate 20. The formulation of any one of claims 1 to 16, further comprising arginine succinate. The formulation of claim 17, wherein the concentration of arginine succinate in the formulation is between about 100 mM and about 300 mM. The formulation of claim 17 or 18, wherein the concentration of the arginine succinate in the formulation is between about 150 mM and about 300 mM. The formulation of any one of claims 17 to 19, wherein the concentration of the arginine succinate in the formulation is between about 150 mM and about 250 mM. The formulation of any one of claims 17 to 20, wherein the concentration of arginine succinate in the formulation is at or about 200 mM. The formulation of any one of claims 1 to 21, further comprising histidine. The formulation of claim 22, wherein the concentration of histidine in the formulation is between about 5 mM and about 40 mM. A formulation as claimed in claim 22 or 23, wherein the concentration of histidine in the formulation is at or about 20 mM. The formulation of any one of claims 1 to 24, wherein the anti-integrin β7 antibody is stable at -20°C for at least about 7 years. The formulation of any one of claims 1 to 25, wherein the anti-integrin β7 antibody is stable at 5°C for at least about 18 months. The formulation of any one of claims 1 to 26, wherein the anti-integrin β7 antibody is stable at 5°C for at least about two years. The formulation of any one of claims 1 to 27, wherein the anti-integrin beta7 antibody is stable at room temperature for at least about 1 day. The formulation of any one of claims 1 to 28, wherein the anti-integrin beta7 antibody is stable at room temperature for up to about 1 month. A formulation comprising an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 Buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, where the concentration of the anti-integrin beta7 antibody is 150 mg/mL or about 150 mg/mL, and wherein the anti-integrin beta7 antibody comprises three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy Chain HVRs: HVR-H1, HVR-H2 and HVR-H3, of which: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) The HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. The formulation of claim 30, wherein the anti-integrin beta7 antibody comprises: a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain variable region comprising SEQ ID NO: 8 The amino acid sequence shown in ID NO: 9. The formulation of claim 30 or 31, wherein the anti-integrin β7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising SEQ ID NO: 11 The amino acid sequence shown in . The formulation of claim 30 or 32, wherein the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO:10; and a heavy chain comprising SEQ ID NO:12 The amino acid sequence shown in . The formulation of any one of claims 30, 32 and 33, wherein the anti-integrin β7 antibody is ilozumab. A formulation comprising an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histidine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody comprises Three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) The HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. The formulation of claim 35, wherein the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 The amino acid sequence shown. The formulation of claim 35, wherein the anti-integrin beta7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence shown. The formulation of any one of claims 35 to 37, wherein the anti-integrin β7 antibody is ilozumab. An article of manufacture comprising the formulation of any one of claims 1 to 38 and a subcutaneous administration device. The article of manufacture of claim 39, wherein the subcutaneous administration device is a needle safety device. The article of claim 39, wherein the needle safety device comprises a prefilled syringe. The article of manufacture of any one of claims 39 to 41, wherein the anti-integrin beta7 antibody is stable at the subcutaneous administration device at 5°C for at least about 60 months, or at 25°C for at least about 3 months . An article of manufacture as claimed in claim 41 or 42, wherein the prefilled syringe comprises a glass barrel, a plunger, a needle and a needle shield or cap. The article of claim 43, wherein the needle shield is a rigid needle shield. The article of claim 44, wherein the rigid needle shield comprises a rubber formulation having a low zinc content. The article of claim 44 or 45, wherein the rigid needle shield comprises an elastic component and a rigid shield. The article of any one of claims 41 to 46, wherein the prefilled syringe is assembled into an auto-injector. The article of any one of claims 40 to 46, wherein the prefilled syringe comprises silicone oil. An article of manufacture as claimed in claim 48, wherein the amount of silicone oil in the prefilled syringe is not greater than about 1 mg. An article of manufacture of claim 48 or 49, wherein the amount of silicone oil in the prefilled syringe is between about 0.1 mg and about 1 mg. An article of manufacture as in any one of claims 48 to 50, wherein the amount of silicone oil in the prefilled syringe is between about 0.2 mg and about 0.6 mg. An article of manufacture as in any one of claims 48 to 50, wherein the amount of silicone oil in the prefilled syringe is between about 0.5 mg and 0.9 mg. An article of manufacture as in any one of claims 40 to 52, wherein the needle safety device has an injection force of not greater than about 50 Newtons (N). An article of manufacture as in any one of claims 40 to 53, wherein the needle safety device has an injection force of not greater than about 35 Newtons (N). The article of manufacture of any one of claims 39 to 54, comprising between about 0.5 mL and about 2.0 mL of the formulation. The article of manufacture of any one of claims 39 to 55, comprising between about 0.5 mL and about 1.0 mL of the formulation. An article of manufacture of any one of claims 39 to 56, comprising about 1.0 mL of the formulation. An article of manufacture of any one of claims 39 to 56, comprising about 0.7 mL of the formulation. The article of manufacture of any one of claims 39 to 55, comprising between about 1.0 mL and about 1.5 mL of the formulation. An article of manufacture of any one of claims 39 to 55 and 59, comprising about 1.4 mL of the formulation. An article of manufacture as claimed in any one of claims 41 to 60, wherein the prefilled syringe has a syringe capacity of 1 mL. An article of manufacture of any of claims 41 to 60, wherein the prefilled syringe has a syringe capacity of 2.25 mL. An article of manufacture comprising about 0.7 mL of a formulation and a subcutaneous administration device, wherein (a) The formulation comprises an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histamine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 acid buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody Comprising three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The hypodermic delivery device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 1 mL. The article of manufacture of claim 63, wherein the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL. An article of manufacture comprising about 1.4 mL of a formulation and a subcutaneous administration device, wherein (a) The formulation comprises an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histamine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 acid buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody Comprising three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The subcutaneous administration device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 2.25 mL. The article of manufacture of claim 65, wherein the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL. An automatic injector comprising the article of any one of claims 39 to 66. An automatic injector comprising an article of manufacture comprising about 0.7 mL of a formulation and a subcutaneous administration device, wherein (a) The formulation comprises an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histamine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 acid buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody Comprising three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The hypodermic delivery device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 1 mL. The auto-injector of claim 68, wherein the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL. An automatic injector comprising an article of manufacture comprising about 1.4 mL of a formulation and a subcutaneous administration device, wherein (a) The formulation comprises an anti-integrin beta7 antibody in 20 mM histidine buffer or about 20 mM histamine at pH 5.8 or between pH 5.7 and 5.9 or between pH 5.75 and 5.85 acid buffer, 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, and wherein the anti-integrin beta7 antibody Comprising three light chain hypervariable regions (HVRs): HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR-H2 and HVR-H3, wherein: (i) the HVR-L1 comprises the amino acid sequence shown in SEQ ID NO:1; (ii) the HVR-L2 comprises the amino acid sequence shown in SEQ ID NO:2; (iii) the HVR-L3 comprises the amino acid sequence shown in SEQ ID NO:3; (iv) the HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4; (v) the HVR-H2 comprises the amino acid sequence SEQ ID NO:5; and (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7, and (b) The hypodermic delivery device is a needle safety device containing a 1 mL prefilled syringe with a syringe capacity of 2.25 mL. The auto-injector of claim 70, wherein the anti-integrin beta7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/mL. A method of treating an inflammatory disorder of the gastrointestinal tract in a subject, comprising administering to the subject an effective amount of a formulation of any one of claims 1 to 38. The method of claim 72, wherein the inflammatory disorder of the gastrointestinal tract is inflammatory bowel disease. The method of claim 73, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. A method of subcutaneously administering a formulation comprising an anti-integrin beta7 antibody, comprising subcutaneously administering the article of manufacture of any one of claims 39 to 66 or the auto-injector of any one of claims 67 to 71 . The method of claim 75, wherein the administering results in reduced or no pain. The method of claim 75 or 76, wherein the administration results in a transient and mild injection site reaction. A method as in any one of claims 75 to 77, wherein the full dose is administered or at least 90% of the full dose is administered. The method of any one of claims 75 to 78, wherein the administering provides an exposure of ilrolizumab equivalent to a prefilled syringe with a needle safety device. The method of any one of claims 75 to 79, comprising the formulation of any one of claims 1 to 37. A formulation as claimed in any one of claims 1 to 38 for use in therapy. An article of manufacture as claimed in any one of claims 39 to 66 for use in therapy. An auto-injector as claimed in any one of claims 67 to 71 for use in therapy. A formulation according to any one of claims 1 to 38, an article of manufacture according to any one of claims 39 to 66, or an auto-injector according to any one of claims 67 to 71, for the treatment of the gastrointestinal tract of a subject Inflammatory disorders of the tract. The formulation, preparation or auto-injector as used in claim 84, wherein the gastrointestinal inflammatory disorder is inflammatory bowel disease. The formulation, preparation or auto-injector as used in claim 85, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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