JP2023536158A - Anti-integrin β7 antibody formulation and device - Google Patents

Abstract

Formulations comprising anti-integrin β7 antibodies or antigen-binding fragments thereof are provided, including pharmaceutical formulations. Articles of manufacture comprising such formulations and methods of using such formulations are also provided. [Selection diagram] Figure 4

Description

Cross-reference to related applications

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

Formulations comprising anti-integrin β7 antibodies or antigen-binding fragments thereof are provided, including pharmaceutical formulations and devices comprising such formulations and methods of using such formulations and devices.
sequence listing

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The name of the ASCII copy made on July 27, 2021 is 00B2061132SL. txt and is 13,697 bytes in size.

Integrins are αβ 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 received widespread interest as potential targets for drug discovery (Sharar et al., Springer Semin. Immunopathol. (1995); 16:359-378). In the immune system, integrins are involved in leukocyte trafficking, adhesion, and infiltration during the inflammatory process (Nakajima et al., J. Exp. Med. (1994); 179:1145-1154). Differential expression of integrins controls 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 expressed primarily on monocytes, lymphocytes, eosinophils, basophils and macrophages, but not on neutrophils (Elices et al., Cell (1990); 60:577-584). The primary ligand for α4β7 integrin is the endothelial surface protein mucosal address in 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 (HEV) at sites of inflammation leads to firm adhesion of leukocytes to the endothelium and subsequent extravasation into the inflamed tissue (Chuluyan et al. al., Springer Semin. Immunopathol., 1995, 16:391404). Monoclonal antibodies against α4β7, MAdCAM or VCAM have been shown to be effective in asthma (Laberge et al., Am. J. Respir. Crit. Care Med. (1995); 151:822-829.), rheumatoid arthritis (Barbadillo et al., Springer Semin. (1995); 16:375-379), colitis (Viney, J. Immunol. (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 β7 antibodies and antigen-binding fragments thereof have been described. See, for example, WO2006/026759. One particular anti-integrin β7 antibody, etrolizumab, is being clinically studied to treat gastrointestinal inflammatory disorders such as inflammatory bowel disease such as ulcerative colitis and Crohn's disease. Results of a phase 1 study of etrolizumab in moderate-to-severe ulcerative colitis were reported by Rutgeerts PJ, et al. Gut 2013;62:1122-1130, reporting that no clinically significant safety signals were observed. Results of a global multicenter phase 2 study of etrolizumab in moderate-to-severe ulcerative colitis showed evidence of clinical efficacy of etrolizumab treatment as measured by induction of clinical remission (Vermeire, S. et al. al., Lancet 2014;384:309-18). Furthermore, clinically meaningful remissions were observed after patients with moderate to severe Crohn's disease were treated with etrolizumab (Sandborn et al., presentation entitled "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). Thus, etrolizumab has demonstrated promise as a therapeutic treatment option in inflammatory bowel disease, and further studies are underway to improve the safety and efficacy profile of etrolizumab.

In each of the previously reported studies, etrolizumab was administered either intravenously or subcutaneously by a healthcare provider in the clinical setting. For subcutaneous administration, vials and syringes with a vial concentration of 150 mg/ml were used. Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are chronic diseases and may require long-term therapeutic treatment with etrolizumab. For optimal patient convenience and compliance, among other advantages, etrolizumab self-administration or home administration by a caregiver or health care professional is desirable. Therefore, it would be advantageous to develop self-administration devices and formulations of etrolizumab that are compatible with such devices.

Because proteins, including antibodies such as etrolizumab, are larger and more complex than traditional organic and inorganic drugs (e.g., with multiple functional groups in addition to complex three-dimensional structures), formulation of such proteins is special. cause problems. For a protein to remain biologically active, the formulation must preserve the integrity of the conformation of at least the core sequence of amino acids of the protein intact while protecting multiple functional groups of the protein from degradation. must. Degradation pathways of proteins can be either chemical instability (e.g. any process that involves modification of proteins by bond formation or cleavage resulting in new chemical entities) or physical instability (e.g. changes in protein conformation). can be accompanied by Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta-elimination or disulfide exchange. Physical instability can result, for example, from denaturation, aggregation, precipitation or adsorption. The three most common proteolytic pathways are protein aggregation, deamidation and oxidation. Cleland et al Critical Reviews in Therapeutic Drug Carrier Systems 10(4):307-377 (1993).

High concentration (e.g., >100 mg/mL) liquid antibody formulations may be used e.g., for routes of therapeutic administration or for therapeutic applications where small amounts of formulation are desired, e.g. preferred for subcutaneous injections containing However, high concentrations of antibody formulations pose many challenges and problems, including those associated with the use of pre-filled syringes or self-administration devices. One problem is instability due to particulate formation. In reconstituted liquid formulations, this problem has been addressed through the use of surfactants (e.g., polysorbate), but surfactants are considered unsuitable for liquid formulations because they make further processing difficult. can be Moreover, surfactants do not further reduce the viscosity build-up caused as a result of the numerous intermolecular interactions from the polymeric nature of the antibody.

The selection of pH and optimal excipients is to prevent particle formation due to polysorbate-induced degradation, to prevent isomerization of certain amino acids and formation of poor intermediates, and to provide advantages for manufacturing. In addition it is important for extended shelf life. Selection of pH and optimal excipients also includes formulation development, e.g. compatibility with storage conditions, pre-filled syringe containing devices such as pre-filled syringes with needle safety devices or auto-injector or self-administration devices. For administration with pre-filled syringes, it is important, for example, to ensure compatibility with device components and to provide low injection forces.

It would be highly advantageous to have formulations comprising anti-β7 antibodies, including etrolizumab, that have long-term stability and low viscosity at high antibody concentrations. High antibody concentration formulations with such properties would be highly advantageous for certain routes of administration, such as subcutaneous administration, including use with pre-filled syringes and self-administration devices. The formulations provided herein address these needs and provide other useful advantages.

It would be highly advantageous to have formulations comprising anti-β7 antibodies with long-term stability and low viscosity at high antibody concentrations. High antibody concentration formulations with such properties would be very advantageous for certain routes of administration, such as subcutaneous administration. The formulations provided herein address these needs and provide other useful advantages.

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

Formulations of the present disclosure comprise, at least in part, the anti-integrin β7 antibody described herein, etrolizumab, formulated in histidine buffer and arginine succinate and detergent at high concentrations (about >100 mg/mL). based on the discovery that such high antibody concentration formulations have low viscosity, extend physical and chemical stability, and maintain potency. The formulations of the present disclosure are optimally suited for self-administration devices such as pre-filled syringes with needle safety devices (PFS-NSD). In certain embodiments, the prefilled syringe is assembled into an autoinjector. Formulations of the present disclosure can be packaged, for example, in subcutaneous administration devices as described herein while maintaining product stability and other desirable attributes. Formulations of the present disclosure are useful, for example, in treating gastrointestinal inflammatory disorders, such as inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease.

Accordingly, in one aspect, formulations are provided comprising an anti-integrin β7 antibody or antigen-binding fragment thereof. In certain embodiments, the concentration of antibody or antigen-binding fragment thereof in the formulation is at least about 100 mg/mL and the viscosity of the formulation is less than about 20 centipoise (cP) at 25°C. In certain embodiments, the viscosity of the formulation is less than about 7 cP at 25°C.

In certain embodiments, the anti-integrin β7 antibody is a monoclonal antibody. In certain embodiments, the anti-integrin β7 antibody is a humanized antibody. In certain embodiments, the anti-integrin β7 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, and
(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;
(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 β7 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 comprising the amino acid sequence set forth in SEQ ID NO:9. . In certain embodiments, the anti-integrin β7 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 the amino acid sequence set forth in SEQ ID NO:11. In certain embodiments, the anti-integrin β7 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 the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the anti-integrin β7 antibody is etrolizumab. In certain embodiments, the concentration of anti-integrin β7 antibody or antigen-binding fragment thereof in the formulation is from about 100 mg/ml to about 220 mg/ml. In certain embodiments, the concentration of anti-integrin β7 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 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 5.6-6.1. In certain embodiments, the pH of the formulation is 5.8, 5.7-5.9, or 5.75-5.85.

In certain embodiments, the formulation comprises arginine-succinate. In certain embodiments, the concentration of arginine succinate in the formulation is from about 100 mM to about 300 mM. In certain embodiments, the concentration of arginine succinate in the formulation is from about 150 mM to about 300 mM. In certain embodiments, the concentration of arginine succinate in the formulation is from about 150 mM to 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 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 0.03% w/v to 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 from about 5 mM to about 40 mM. In certain embodiments, the concentration of histidine in the formulation is at or about 20 mM.

In certain embodiments, the formulation has long-term stability. In certain embodiments, the anti-integrin β7 antibody is stable at -20°C for at least about 7 years. In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof is stable at 5° C. for at least about 1 year. In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof is stable at 5° C. for at least about 5 years. In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof is stable at 5° C. for about 6 years.

In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof is stable at room temperature for at least about 1 day. In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof is stable at room temperature for up to about 1 month.

In another aspect, the disclosure provides a formulation comprising an anti-integrin β7 antibody or antigen-binding fragment thereof in 20 mM or about 20 mM histidine buffer (pH 5.8), 0.04% polysorbate 20 and 200 mM or about 200 mM arginine succinate. and the concentration of the anti-integrin β7 antibody is about 150 mg/ml, and the anti-integrin β7 antibody comprises three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2 and HVR-L3, and three heavy chain HVR, HVR-H1, HVR-H2 and HVR-H3;
(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;
(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 of 5.7-5.9 or 5.75-5.85. In certain embodiments, the formulation comprises 0.04% polysorbate 20 or about 0.04% polysorbate 20.

The subject of the present disclosure is 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20. Formulations are provided comprising an anti-integrin β7 antibody in 04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, the anti-integrin β7 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, (i) HVR-L1 comprising 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, and (iv) HVR-H1 comprises the amino acid sequence shown in SEQ ID NO:4. (v) HVR-H2 comprises the amino acid sequence shown in SEQ ID NO:5; (vi) said HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. In certain embodiments, the anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11. In certain embodiments, the anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the anti-integrin β7 antibody is etrolizumab.

In certain embodiments, the anti-integrin β7 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 comprising the amino acid sequence set forth in SEQ ID NO:9. . In certain embodiments, the anti-integrin β7 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 the amino acid sequence set forth in SEQ ID NO:11. In certain embodiments, the anti-integrin β7 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 the amino acid sequence set forth in SEQ ID NO:12.

In a still further aspect, an article of manufacture is provided comprising a subcutaneous administration device. In certain embodiments, the subcutaneous administration device delivers a flat dose of an anti-integrin β7 antibody or antigen-binding fragment thereof to the 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 β7 antibody is etrolizumab. The anti-integrin β7 antibody or antigen-binding fragment thereof in the subcutaneous administration device is formulated as described above to provide 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 pre-filled syringe. In certain embodiments, the anti-integrin β7 antibody is stable in a subcutaneous administration device at 5° C. for at least about 60 months or at 25° C. for at least about 3 months. In certain embodiments, the pre-filled syringe comprises a glass barrel, plunger stopper, needle, and needle shield or tip cap. In certain embodiments, the needle shield is a rigid needle shield. In certain embodiments, the rigid needle shield comprises a low zinc content rubber compound. In certain embodiments, a rigid needle shield includes an elastomeric component and a rigid shield. In certain embodiments, the prefilled syringe is assembled into an autoinjector.

In certain embodiments, the volume of formulation contained in the pre-filled syringe is from about 0.5 mL to about 2.0 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is from about 0.5 mL to about 1.0 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 0.7 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 0.75 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.0 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is from about 1.0 mL to about 1.5 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.4 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.5 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.45 mL. In certain embodiments, the pre-filled syringe has a syringe capacity of 1 mL. In certain embodiments, the pre-filled syringe has a syringe capacity of 2.25 mL.

In certain embodiments, the pre-filled syringe comprises silicone oil. In certain embodiments, the amount of silicone oil in the pre-filled syringe is about 1 mg or less. In certain embodiments, the amount of silicone oil in the pre-filled syringe is from about 0.1 mg to about 1 mg. In certain embodiments, the amount of silicone oil in the pre-filled syringe is from about 0.2 mg to about 0.6 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is about 0.5 mg to 0.9 mg.

In certain embodiments, the needle safety device has an injection force of about 50 Newtons (N) or less. In certain embodiments, the needle safety device has an injection force of about 35 Newtons (N) or less. In certain embodiments, the needle safety device has an injection force of about 33 Newtons (N) or less.

The presently disclosed subject matter provides an article of manufacture comprising about 0.7 mL formulation and a subcutaneous administration device,
(a) The formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20. 04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, comprising an anti-integrin β7 antibody comprising three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2 and comprising HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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;
(vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;
(b) The subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe with a 1 mL syringe capacity.

In certain embodiments, the anti-integrin β7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/ml.

The presently disclosed subject matter provides an article of manufacture comprising about 1.4 mL of formulation and a subcutaneous administration device,
(a) The formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20. 04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, comprising an anti-integrin β7 antibody comprising three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2 and comprising HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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;
(vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;
(b) The subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe with a syringe capacity of 2.25 mL.

In certain embodiments, the anti-integrin β7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/ml.

The presently disclosed subject matter further provides automatic injection devices including the articles of manufacture disclosed herein.

The presently disclosed subject matter provides an auto-injection device comprising an article of manufacture comprising about 0.7 mL of formulation and a subcutaneous administration device,
(a) The formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20. 04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, comprising an anti-integrin β7 antibody comprising three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2 and comprising HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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;
(vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;
(b) The subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe with a 1 mL syringe capacity.

In certain embodiments, the anti-integrin β7 antibody is present in the formulation at a concentration of 150 mg/mL or about 150 mg/ml.

The presently disclosed subject matter provides an autoinjector device comprising an article of manufacture comprising about 1.4 mL of formulation and a subcutaneous administration device,
(a) The formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20. 04% polysorbate 20, and 200 mM arginine succinate or about 200 mM arginine succinate, comprising an anti-integrin β7 antibody comprising three light chain hypervariable regions (HVRs), HVR-L1, HVR-L2 and comprising HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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;
(vi) HVR-H3 comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;
(b) The subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe with a syringe capacity of 2.25 mL.

In certain embodiments, the anti-integrin β7 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 a gastrointestinal inflammatory disorder in a subject is provided. In certain embodiments, the method comprises administering to the subject an effective amount of any of the above formulations.

In yet another aspect, a method of subcutaneously administering a formulation comprising an anti-integrin β7 antibody or antigen-binding fragment thereof is provided. Such methods include subcutaneous administration of any of the formulations described above. In certain embodiments, the method includes a subcutaneous administration device according to any of the devices described above. In certain embodiments, the method includes an autoinjector disclosed herein. In certain embodiments, administration results in mild pain or no pain. In certain embodiments, administration results in a transient and mild injection site reaction. In certain embodiments, the entire dose is administered, or at least 90% of the total dose is administered. In certain embodiments, administration provides equivalent exposure to etrolizumab compared to a pre-filled syringe with a needle safety device.

The presently disclosed subject matter also provides the use of the formulations, articles of manufacture, or autoinjection devices disclosed herein in therapy.

Further, the subject matter disclosed herein provides use of the formulations, articles of manufacture, or auto-injection devices disclosed herein in treating gastrointestinal inflammatory disorders 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.

1 shows the solubility curves of lauric acid, myristic acid and palmitic acid as a function of pH and polysorbate 20 (PS20) concentration as described in Example 1. FIG. FIG. 2 shows the effect of arginine on solution viscosity, as described in Example 1. 3 shows the effect of protein concentration on viscosity of formulations containing 200 mM arginine succinate, as described in Example 1. FIG. 4 shows the change in the percentage of high molecular weight species (HMWS) of etrolizumab in pre-filled syringes containing varying amounts of silicone oil, as described in Example 1. FIG. The terms "high molecular weight species (HMWS)" and "high molecular weight form (HMWF)" are used interchangeably herein. 5 shows the percentage of HMWS of etrolizumab in various concentrations of tungsten over time, as described in Example 1. FIG. FIG. 6 shows the effect of zinc on the viscosity of etrolizumab at various protein concentrations in formulations containing 20 mM histidine, 200 mM arginine succinate, pH 5.8 at 25° C., as described in Example 1. FIG. 7 shows protein aggregate formation by 50 mM zinc and 150 mg/mL etrolizumab at room temperature, as described in Example 1. FIG. 8 shows HMWS formation by 10 mM zinc and 10 mg/mL or 50 mg/mL etrolizumab at 40° C., as described in Example 1. FIG. 9 shows the effect of histidine on HMWS formation in formulations containing 10 mM zinc and 10 mg/mL etrolizumab at 40° C., as described in Example 1. FIG. 10 shows the effect of succinate on HMWS formation in formulations containing 10 mM zinc and 10 mg/mL etrolizumab at 40° C., as described in Example 1. FIG. 11 shows various concentrations of histidine and succinate, and the combined effect on HMWS formation in formulations containing 10 mM zinc and 50 mg/mL etrolizumab at 40° C., as described in Example 1. show. FIG. 12 shows an exemplary pre-filled syringe (top 2) and autoinjector (bottom) as described in Example 2. 13 shows a prefilled autoinjector for etrolizumab as described in Example 2. FIG. FIG. 14 shows the autoinjector (AI) tolerability and human factor study design as described in Example 2. 15 shows a graph showing pain over time by intensity (7-point visual descriptive scale), as described in Example 2. FIG. 16 shows a graph showing pain by injection site over time (7-point visual descriptive scale), as described in Example 2. FIG. FIG. 17 shows a two-part pharmacokinetic bridging study design as described in Example 2. * Justifying a Geometric Mean Ratio (GMR) of 1.15 as a decision point is likely that a 15% difference between needle-safe pre-filled syringes (PFS-NSD) is realistic and therefore , this study was based on the assumption that bioequivalence cannot be demonstrated. ^† Adjustments to GMR-driven N from pilot studies. AI autoinjector; AUC area under the curve; C _max maximum serum concentration of etrolizumab. FIG. 18 shows the placement of participants as described in Example 3. FIG. AI autoinjector, area under the AUC curve, PFS-NSD prefilled syringe with needle safety, PK pharmacokinetics, SC subcutaneous. ^a Excluded due to eligibility criteria (weight limit). Participants were excluded from certain PK analyzes due to insufficient PK data for calculation. 2 shows the effect of body weight on etrolizumab C _max (top) or AUC _0-inf (bottom) as described in Example 3. FIG. AI autoinjector, area under the AUC curve, AUC _0-inf AUC extrapolated to infinity, C _max maximum concentration, PFS-Prefilled syringe with NSD needle safety, SC subcutaneous. FIG. 20 shows etrolizumab serum concentrations over time using AI and PFS-NSD on linear (A) and semi-logarithmic (B) scales in a pivotal study as described in Example 3. AI autoinjector, pre-filled syringe with PFS-NSD needle safety. FIG. 21 shows AI (N=73 total, 20 subjects ADA positive, top) and PFS-NSD (N=75, 24 subjects ADA positive, bottom) as described in Example 3. ) shows etrolizumab serum concentration over time by ADA status using . ADA anti-drug antibody, AI autoinjector, pre-filled syringe with PFS-NSD needle safety. FIG. 22 shows definitions of various forces during injection as described in Example 4.

Unless defined otherwise, 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 ed. , J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed. , John Wiley & Sons (New York, NY 1992) provides those skilled in the art with a general guide to many of the terms used in this application.

Certain Definitions For the purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall include the plural and vice versa. Terms also include the singular. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" do not explicitly dictate otherwise. As long as it includes more than one referent. Thus, for example, reference to a "protein" or "antibody" includes multiple proteins or antibodies, respectively, reference to "a cell" includes mixtures of cells, and the like.

As used herein, the terms "about" or "approximately" mean an acceptable margin of error for a particular value as determined by one skilled in the art, which is how that value is measured or determined. or depends in part on the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations per practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude, preferably within five times, more preferably within two times the value. Any reference herein to a value or parameter following “about” includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X."

The term "pharmaceutical formulation" refers to a preparation that is in such a form as to render the biological activity of the active ingredients effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. . Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be administered to a subject mammal in moderation to provide an effective dose of the active ingredient employed.

A "sterile" formulation is sterile or free or essentially free of all viable microorganisms and their spores.

A "frozen" formulation is a formulation below 0°C. Generally, frozen formulations are not freeze-dried and are not pre- or post-lyophilized. In some embodiments, the frozen formulation comprises frozen drug for storage (in stainless steel tanks) or frozen medical product (in final vial configuration).

A "stable" formulation is one in which the protein therein essentially retains its physical and/or chemical stability and/or biological activity upon storage. In certain embodiments, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation.

As used herein, a formulation with "long-term stability" is defined as the physical stability, chemical stability, or , and formulations that essentially retain biological activity. In certain embodiments, storage is at 5° C. for 1 year or more. In certain embodiments, storage is at 5° C. for up to 5 or 6 years. In certain embodiments, the anti-integrin β7 antibody is stable at room temperature for at least about 1 day. In certain embodiments, the anti-integrin β7 antibody is stable at room temperature for up to about 1 month. As used herein, room temperature is from about 20°C to about 22°C. In certain embodiments, the room temperature is about 20°C.

If the protein shows little or no signs of aggregation, precipitation, and/or denaturation upon visual inspection for color and/or clarity, or as measured by UV light scattering or size exclusion chromatography, "Retains its physical stability" in a pharmaceutical formulation.

A protein is defined as "its Retains chemical stability'. Chemical stability can be assessed by detecting and quantifying chemically modified forms of the protein. Chemical modification may include resizing (e.g. clipping), assessed using e.g. size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS). can do. Other types of chemical alterations include charge alterations (eg, resulting from deamidation) that can be assessed by, eg, ion-exchange chromatography or imaging capillary isoelectric focusing (icIEF).

Antibodies have approximately the biological activity exhibited at the time the pharmaceutical formulation is prepared, when the biological activity of the antibody at a given time is determined, for example, in an antigen binding assay or a potency assay. "Retains its biological activity" in a pharmaceutical formulation if it is within 20% (within the error of the assay).

As used herein, "biological activity" of a monoclonal antibody refers to the antibody's ability to bind to an antigen. It can further include antibodies that bind antigens and produce a measurable biological response that can be measured in vitro or in vivo. Such activity can be antagonist activity or agonist activity.

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

By "isotonic" is meant that the intended formulation has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freeze-type osmometer.

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

As used herein, "surfactant" refers to surfactants, typically nonionic surfactants. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; betaine, myristylsulfobetaine, linoleylsulfobetaine, or stearylsulfobetaine; laurylsarcosine, myristylsarcosine, linoleylsarcosine, or stearylsarcosine; linoleylbetaine, myristylbetaine, or cetylbetaine; lauroamidopropylbetaine, cocamidopropylbetaine , linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyldimethylamine, palmidopropyldimethylamine, or isostearamidopropyldimethylamine; methyl cocoyl sodium taurate or disodium methyl oleyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl glycol, and ethylene glycol-propylene glycol copolymers such as Pluronics, PF68, etc.) and the like. In certain embodiments, the surfactant is polysorbate 20.

As used herein, "treatment" refers to clinical intervention aimed at altering the natural history of the individual or cells being treated, and can occur prior to or during the clinical pathological process. . Desirable effects of treatment include preventing the onset or recurrence of the disease or a condition or symptom thereof, alleviating the condition or symptoms of the disease, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, Improvement or alleviation of the condition and remission or improved prognosis are included.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent may vary according to factors such as the individual's medical condition, 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 human.

A "medicine" is an active drug for treating a disease, disorder, and/or condition.

"Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins with similar structural characteristics. Although antibodies exhibit binding specificity for a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. Polypeptides of the latter type, for example, are 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 (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies ( For example, they include bispecific antibodies) and may also include specific antibody fragments (as described in more detail herein), so long as they exhibit the desired biological activity. Antibodies can be chimeric human antibodies, humanized antibodies, and/or affinity matured antibodies.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to antibodies in their substantially intact form, other than the antibody fragments described below. be. These terms specifically refer to antibodies with heavy chains that include an Fc region.

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

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named to reflect its ability to readily crystallize. is produced. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. Collectively, the six CDRs of Fv confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) lacks the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site. have.

The Fab fragment contains the heavy and light chain variable domains and also contains the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the constant domain cysteine residue(s) bear a free thiol group. F(ab') ₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., individual antibodies that make up the population may be present in small amounts. are identical except for minor mutations, eg, naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically comprise antibodies comprising a target-binding polypeptide sequence, wherein the target-binding polypeptide sequence comprises a single target-binding sequence from a plurality of polypeptide sequences. It was obtained by a process involving the selection of polypeptide sequences. For example, the selection process can be the selection of unique clones from a plurality of clones such as hybridoma clones, phage clones, or pools of recombinant DNA clones. The selected target binding sequence e.g. improves affinity to the target, humanizes the target binding sequence, improves its production in cell culture, reduces its immunogenicity in vivo, multispecificity It is understood that antibodies that may be further modified to produce antibodies and that contain modified target binding sequences are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by, for example, hybridoma methods (see, eg, Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 ⁽ Elsevier, NY, 1981)), recombinant DNA methods (see, eg, U.S.A., 2nd ed. 1988); S. Patent No. 4, 816, 567), phage display technologies (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); 1093 (2004);Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472 (2004); 2004)) and techniques for the production of human or human-like antibodies in animals that possess some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 98/ WO 96/33735; WO 91/10741; Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551 (1993); al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al. , Bio. Technology 10:779-783 (1992); Lonberg et al. , Nature 368:856-859 (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 a portion of the heavy and/or light chain with corresponding sequences in an antibody from a particular species or belonging to a particular antibody class or subclass. "Chimeric" antibodies that are identical or homologous, while the remainder of the chain(s) are identical or homologous to corresponding sequences in an antibody from another species or belonging to another antibody class or subclass , and fragments of such antibodies so long as they exhibit the desired biological activity (U.S. 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 varying structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by one constant light (CL) domain. The light chains of antibodies can be assigned to one of two classes, called kappa and lambda, based on the amino acid sequences of their constant domains.

The terms "variable region" or "variable domain" refer to the domains of the heavy or light chains of an antibody that are involved in binding the antibody to antigen. The variable domains of the heavy and light chains of naturally occurring antibodies ( _VH and _VL , respectively) have a generally similar structure, each domain consisting of four conserved framework regions (FR), It contains three hypervariable regions (HVRs). (See, eg, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single V _H or V _L domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using a _VH or _VL domain of the antigen-binding antibody _{and screening a library of complementary VL or VH domains} , respectively. good too. For example, Portolano et al. , J. Immunol. 150:880-887 (1993); Clarkson et al. , Nature 352:624-628 (1991).

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to non-human antibodies. and all or substantially all of the FRs correspond to a human antibody. A humanized antibody optionally may 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 antibody variable regions that are hypervariable in sequence and/or form structurally defined loops. Refers to an area of a domain. Generally, antibodies contain six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several hypervariable region descriptors have been used and are included 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)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM hypervariable regions are a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" hypervariable regions are based on analysis of available complex crystal structures. Residues from each of these HVRs are shown below.

Loop Kabat AbM Chothia Contact 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 are the following "extended hypervariable regions": 24-36 or 24-34 in the VL (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) within VH, and 93-102, 94-102, or 95-102 (H3). Variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

Depending on the amino acid sequences of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes), e.g., _IgG1 , _IgG2 , _IgG3 , IgG4 _. , IgA ₁ , and IgA ₂ . The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional organization of various classes of immunoglobulins are well known, see, for example, 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 region possessed by its heavy chains. Antibodies are divided into five major classes: IgA, IgD, IgE, IgG and IgM, some of which are further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgAl and IgA2. can be done. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, 8, y, and μ, respectively.

An "isolated" biological molecule, such as a 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.

A "subcutaneous administration device" refers to a device adapted or designed to administer a drug, eg, therapeutic antibody, or pharmaceutical formulation, by a subcutaneous route. Exemplary subcutaneous administration devices include injection devices, including needle safety devices (e.g., with pre-filled syringes), auto-injection devices (e.g., with pre-filled syringes), infusion pumps, pen injectors, needle-free Devices, and patch delivery systems include, but are not limited to. The subcutaneous administration device dispenses a fixed volume of 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, about 1.25 mL, about 1.4 mL, about 1.5 mL, about 1.75 mL, about 2.5 mL, about 1.25 mL, about 1.4 mL, about 1.5 mL, about 1.75 mL, about 2.0 mL. Administer 0 mL or approximately 5.0 mL.

A "package insert" or "label" includes information about the indications, directions for use, dosage, administration, contraindications, other therapeutic agents with which the packaged product is combined, and/or the use thereof, of such therapeutic or medicinal product. Used to refer to the instructions customarily included in commercial packages of therapeutic or pharmaceutical products containing warnings about

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

"Inflammatory bowel disease" or "IBD" are used interchangeably herein to refer to bowel diseases that cause inflammation and/or ulceration and include, but are 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. Crohn's disease, unlike ulcerative colitis, can affect any part of the intestine. The most prominent feature of Crohn's disease is a granular, reddish-purple, edematous thickening of the intestinal wall. With the development of inflammation, these granulomas often lose their circumscribing boundaries and integrate with the surrounding tissue. Diarrhea and intestinal obstruction are prominent clinical features. Like ulcerative colitis, the course of Crohn's disease can be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease is characterized by an intestinal It is not curative by excision of the area involved. Most Crohn's disease patients require surgery at some point, but later relapses are common and ongoing medical treatment is the norm.

Crohn's disease can affect any part of the gastrointestinal tract from the mouth to the anus, but typically presents in the ilecolon, small intestine, or colon-anorectal region. Histopathologically, the disease is manifested by granulomatoma, crypt abscesses, fissures, and aphthous ulcers. There is a mixed inflammatory infiltrate consisting of lymphocytes (both T and B cells), plasma cells, macrophages and neutrophils. There is a disproportionate increase in IgM- and IgG-secreting plasma cells, macrophages, and neutrophils. The anti-inflammatory drugs sulfasalazine and 5-aminosalicylic acid (5-ASA) are useful in treating mildly active Crohn's disease of the colon and are commonly prescribed to maintain remission of the disease. Metroidazole and ciprofloxacin are similar in efficacy to sulfasalazine and appear to be particularly useful for treating perianal disease. In more severe cases, corticosteroids are effective in treating active exacerbations and can even maintain remission. Azathioprine and 6-mercaptopurine have also shown success in patients requiring long-term administration of corticosteroids. It is also possible that these drugs play a role in long-term prevention. Unfortunately, in some patients there can be a very long delay (up to 6 months) before onset of action.

Anti-diarrheal drugs can also provide symptomatic relief in some patients. Nutritional therapy or elemental nutrition can improve a patient's nutritional status and induce amelioration of symptoms of acute illness, but does not induce sustained clinical remission. Antibiotics are used to treat secondary small intestinal bacterial overgrowth and to treat suppurative complications.

"Ulcerative colitis (UC)" affects the large intestine. The course of the disease may be continuous or relapsing, mild or severe. The earliest lesion is an inflammatory infiltrate with abscess formation at the base of the Lieberkuhn crypts. The coalescence of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply and lead to ulceration. Symptoms of this disease include cramping, lower abdominal pain, rectal bleeding, and frequent loose stools consisting mainly of blood, pus and mucus, with few fecal particles. Acute, severe, or chronic unrelenting ulcerative colitis may require total colectomy. The clinical features of UC are highly variable, onset can be insidious or acute and can include diarrhea, tenesmus and recurrent rectal bleeding. Fatal involvement of the entire colon can result in toxic colectomy, a life-threatening emergency. 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. Typically, whole blood includes all blood components, such as cellular components and plasma. Methods for obtaining whole blood from mammals are well known in the art.

Therapeutic Agents Provided herein are therapeutic agents for treating gastrointestinal inflammatory disorders (eg, inflammatory bowel disease). Inflammatory bowel disease (IBD) severely impacts patient 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 main forms of IBD are ulcerative colitis (UC) and Crohn's disease, two distinct conditions that share some common symptoms and exhibit partially overlapping etiologies (Zhang and Li, World J Gastroenterol. 2014;20(1):91-9;Abraham and Cho, N Engl J Med.2009;361(21):2066-78.Current pharmacologic therapies for IBD are not curative. Many pharmacological therapies for can lose efficacy over the duration of the disease and lead to systemic side effects (Abraham and Cho, N Engl J Med. 2009;361(21):2066-78; Rogler, Best Pract Res Clin Gastroenterol.2010;24(2):157-65).Etrolizumab is an anti-β7 integrin monoclonal antibody under development for patients with UC and Crohn's disease.Etrolizumab selectively inhibits α4β7 and αEβ7. (Zundler et al., Gut. 2019;68(9):1688-700).The efficacy of etrolizumab in patients with UC and Safety 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 β7 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-integrin β7 antibody is a humanized monoclonal anti-integrin β7 antibody. In certain such embodiments, the anti-integrin β7 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, and (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 sequence (vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7;

In certain such embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:9. and a region domain. In certain embodiments, the anti-integrin β7 antibody or antigen-binding fragment thereof comprises a light chain variable region domain comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12 and a heavy chain comprising the amino acid sequence shown in In certain embodiments, the anti-integrin β7 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 domain comprising the amino acid sequence set forth in SEQ ID NO:9.

In certain such embodiments, the anti-integrin β7 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 the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12 and a heavy chain comprising the amino acid sequence shown in In certain such embodiments, the anti-integrin β7 antibody is etrolizumab. SEQ ID NOs: 1-12 are provided below. Anti-integrin β7 antibodies or antigen-binding fragments thereof are further described in WO 2006/026759, incorporated herein by reference.

Certain Molecular Biomarkers In certain instances, biomarkers are quantified in biological samples obtained from subjects as a means of selecting subjects for treatment with a given therapeutic agent. International Patent Publication Nos. WO2014160753, WO2015148809 and WO2009140684 disclose methods of predicting responsiveness of patients with gastrointestinal inflammatory disorders to anti-integrin β7 antibody formulations described herein, and Methods of selecting subjects with gastrointestinal inflammatory disorders for treatment with anti-integrin β7 antibody formulations described herein are described.

General Techniques for Formulation Formulations containing anti-integrin β7 antibodies or antigen-binding fragments thereof can be prepared as further described herein using certain excipients and techniques known in the art. can be prepared and analyzed. 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 the need to address issues not encountered with full-length antibodies (e.g., clipping of antibody to Fab). can occur. The therapeutically effective amount of antibody present in the formulation is determined by taking into account the desired dose volume and mode(s) of administration. In certain embodiments, from about 0.1 mg/mL to about 250 mg/mL, or from about 10 mg/mL to about 220 mg/mL, or from about 50 mg/mL to about 220 mg/mL, or from about 100 mg/mL to about 220 mg/mL , or from about 100 mg/mL to about 150 mg/mL, or from about 150 mg/mL to about 200 mg/mL are exemplary antibody concentrations in the formulation. In certain embodiments, anti-integrin β7 antibodies are formulated at a concentration of 150 mg/mL.

An aqueous formulation is prepared comprising an anti-integrin β7 antibody or antigen-binding fragment thereof in a pH buffered solution. The buffer can have a pH ranging from 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 above 5.5. In certain embodiments, the pH is 5.5-6.1. In certain embodiments, the pH is 5.6-6.1. 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 5.7-5.9. In certain embodiments, the pH is 5.75-5.85. The pH of the formulations of the present disclosure is higher than standard antibody formulations with similar excipient compositions. While typical antibody formulations have a pH of 5.5, the disclosed formulations presented have a pH greater than 5.5, such as 5.8, 5.7-5.9 or 5.75-5.85. has a pH of A higher formulation pH reduces the risk of particle formation as a result of polysorbate degradation during long-term storage in high protein concentration pre-filled syringes. Increased solubility of free fatty acids at higher pH, which may be due to polysorbate degradation, reduces the risk of particle formation. A pH greater than 5.5, such as a pH of 5.8, balances the risk of particle formation with the chemical and physical stability of the antibody. A pH greater than 5.5, such as a pH of 5.8, minimizes the rate of Asp isomerization and succinimide intermediate formation, which substantially affects antibody chemical stability and thereby product quality. Allows for patient convenience in combination with the device by allowing storage at ambient temperature without feeding.

Examples of buffers that control the pH within this range include acetates (e.g. histidine acetate, arginine acetate, sodium acetate), succinates (e.g. histidine succinate, arginine succinate, sodium succinate), gluconate Acid, citrate and other organic acid buffers and combinations thereof. Buffer concentrations can be, for example, from about 1 mM to about 600 mM, depending on the buffer and the desired isotonicity of the formulation. In certain embodiments, the buffer contains histidine. The presence of histidine in formulations can significantly reduce the rate of high molecular weight species (HMWS) formation in the presence of zinc. The concentration of histidine in the formulation is about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 15 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM. can be 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 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 from about 150 mM to about 200 mM. In certain embodiments, the concentration of arginine succinate in the formulation is about 200 mM. High arginine concentrations and high conductivity formulations shield the charge on the antibody and prevent pH shifts. In addition, arginine can affect the viscosity of formulations, eg viscosity of formulations 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 viscosity of the formulation is from about 1 cP to about 20 cP at 25°C, from about 5 cP to about 20 cP at 25°C, from about 5 cP to about 15 cP at 25°C, from about 1 cP to about 10 cP at 25°C, or from about 5 cP to about 10 cP. In certain embodiments, the formulation has a viscosity of about 7 cP at 25°C.

In certain embodiments, an 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.), pluronic polyols, Triton®, polyoxyethylene sorbitan monoether (Tween (registered trademark)-20, Tween (registered trademark)-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic surfactants that may be used include sodium lauryl sulfate, sodium dioctylsulfosuccinate and sodium dioctylsulfonate. Cationic surfactants include benzalkonium chloride or benzethonium chloride. In certain embodiments, the surfactant is polysorbate 20.

Non-ionic surfactants not only help solubilize the therapeutic agent, but can also help protect the therapeutic protein from agitation-induced aggregation, which can also help protect the therapeutic protein from the active therapeutic protein or antibody. Allows the formulation to be exposed to shear surface stress without causing denaturation.

The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of microparticles in the formulation and/or reduces adsorption. For example, surfactants may be present in the formulation in amounts 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 is about 0.005% w/v to about 0.5% w/v, about 0.02% w/v to about 0.5% w/v, about 0.03 % w/v to about 0.5% w/v, 0.03% w/v to 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 of the present disclosure contain 0.04% w/v polysorbate 20. Higher concentrations of polysorbate 20 help solubilize free fatty acids that may be produced as a result of polysorbate breakdown, thereby reducing the risk of forming particles.

In certain embodiments, the formulation comprises an agent as defined above (e.g., antibodies, buffers, and surfactants) and one such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium Cl. Essentially free of the above preservatives. In certain embodiments, the formulation is free of preservatives. In certain embodiments, a preservative may be included in the formulation, particularly the formulation is a multiple dose formulation. The concentration of preservatives can range from about 0.1% to about 2%, such as from about 0.5% to about 1%. one or more other pharmaceutically acceptable carriers, excipients, or stabilizers, see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A.; Ed. (1980) may be included in the formulation, provided that they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: additional 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 counterions.

Although various descriptions of chelating agents herein often focus on EDTA, it will be appreciated that other metal ion chelating agents are also encompassed by the present invention. Metal ion chelating agents are well known to those skilled in the art and include aminopolycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(β-aminoethylether)-N,N,N', N′-tetraacetic acid), NTA (nitrilotriacetic acid), EDDS (ethylenediamine disuccinate), PDTA (1,3-propylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), ADA (β-alanine diacetic acid), MGCA (methylglycine diacetic acid) and the like, but are not necessarily limited to these. Additionally, some embodiments herein include phosphonate/phosphonic acid chelators.

Tonicity agents, sometimes known as "stabilizers," are present to adjust or maintain the isotonicity of liquid compositions. When used with large charged biomolecules such as proteins and antibodies, tonicity agents can interact with charged groups on amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. They are often called "stabilizers". The tonicity agent can be present in an amount of 0.1% to 25% by weight, or 1% to 5%, taking into account the relative amounts of the other ingredients. Tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional stabilizers include a wide range of excipients that range in function from bulking agents to solubility enhancers to agents that prevent denaturation or adhesion to container walls. Stabilizers can be present in the range of 0.1 to 10,000 parts by weight of active protein or antibody. Typical stabilizers include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, 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, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitol (e.g., inositol ), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin. or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (eg xylose, mannose, fructose, glucose); disaccharides (eg lactose, maltose, sucrose); trisaccharides such as raffinose; For example dextrin or dextran.

Various analytical techniques for measuring protein stability are available in the art, eg, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed. , Marcel Dekker, Inc.; , New York, N.L. Y. , Pubs. (1991) and Jones, A.; Adv. Drug Delivery Rev. 10:2990 (1993). Stability can be measured at a selected temperature for a selected period of time. In certain embodiments, the formulation is stable at about 40° C. for at least about 1 week. In certain embodiments, the formulation is stable at about 5°C for at least about 12 months, and/or is stable at about 5°C for at least about 18 months, and/or is stable 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 formulation is stable at about -20°C for at least 2 years, and/or is stable at about -20°C for at least 4 years, and/or is stable at about -20°C for at least about 5 years. 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 formulation is stable at about 25°C for at least about 1 week and/or stable at about 25°C for at least about 2 weeks, or stable at about 25°C for at least about 4 weeks. In certain embodiments, the formulation is stable after freezing (eg, to −70° C.) and thawing the formulation, eg, after 1, 2, 3, 4, or 5 cycles of freezing and thawing. Stability is assessed by aggregate formation (e.g. assessed using size exclusion chromatography, assessed by turbidity measurements and/or assessed by visual inspection); cation exchange chromatography, image capillary isoelectric focusing (e.g. icIEF) or by assessing charge heterogeneity using capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; It can be assessed qualitatively and/or quantitatively in a wide variety of ways, including, for example, trypsin or LYS-C) assays; assessing the biological activity or antigen-binding function of the antibody, and the like. Instability includes aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation , unpaired cysteines, N-terminal extensions, C-terminal processing, glycosylation changes, and the like.

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

In certain embodiments, an anti-integrin β7 antibody or antigen-binding fragment thereof is administered, eg, using an autoinjector, autoinjector, or other device designed for self-administration. In certain embodiments, an anti-integrin β7 antibody or antigen-binding fragment thereof is administered using a subcutaneous administration device. Various self-injection and subcutaneous administration devices, including auto-injection devices, are known in the art and commercially available. Exemplary devices include prefilled syringes (BD HYPAK SCF®, BD NEOPAK™, READYFILL™ and STERIFILL SCF™ from Becton Dickinson; CLEARSHOT™ copolymer prefilled from Baxter). syringes; and Daikyo Seiko CRYSTAL ZENITH® pre-filled syringes available from West Pharmaceutical Services); disposable pen injection devices such as the BD Pen manufactured by Becton Dickinson; INJECT-EASE™ and microinfuser devices; and H-PATCH™ from Valeritas, etc.), and needle-free injection devices (BIOJECTOR® and IJECT® available from Bioject; and SOF-SERTER® and patch devices available from Medtronic), but not limited to. Certain embodiments of subcutaneous administration devices are further described herein. Co-formulation or co-administration of an anti-integrin β7 antibody or antigen-binding fragment thereof and at least a second therapeutic compound with such an autoinjector or subcutaneous administration device is envisioned.

Recombinant Methods Antibodies may be produced using recombinant methods and compositions described, for example, in US Pat. No. 4,816,567. In certain embodiments, nucleic acid molecules are provided that encode an anti-integrin β7 antibody or antigen-binding fragment thereof described herein. Such nucleic acid molecules may encode the amino acid sequences comprising the _VL of the antibody and/or the amino acid sequences making up the _VH of the antibody (eg, the light and/or heavy chains of the antibody). In certain embodiments, one or more vectors (eg, expression vectors) containing such nucleic acid molecules are provided. In certain embodiments, host cells containing such nucleic acid molecules are provided. In certain embodiments, the host cell comprises (e.g., transformed): (1) a nucleic acid molecule encoding an amino acid sequence comprising the _VL of the antibody; and an amino acid sequence comprising the _VH of the antibody. or (2) a first vector comprising a nucleic acid molecule encoding an amino acid sequence comprising the _VL of the antibody and a second vector comprising a nucleic acid molecule encoding an amino acid sequence comprising the _VH of the antibody A vector containing and . In certain embodiments, host cells are eukaryotic cells such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells). In a specific embodiment, a method of making an anti-integrin β7 antibody comprising culturing a host cell comprising a nucleic acid molecule encoding an antibody provided above under conditions suitable for expression of the antibody; optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-integrin β7 antibodies, nucleic acid molecules encoding the antibodies, eg, as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acid molecules are readily isolated using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). can be analyzed and sequenced.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly if they do not require glycosylation or Fc effector functions. See, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003) describing expression of antibody fragments in E. coli, pp. 245 -254). After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and further purified.

In addition to prokaryotes, eukaryotes such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including strains and yeast in which the glycosylation pathway has been "humanized." Strains are included that produce antibodies with a partially or fully human glycosylation pattern. Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al. , Nat. Biotech. 24:210-215 (2006).

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

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

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 strain transformed with SV40 (COS-7); (1977))); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); African Green Monkey Kidney Cells (VERO-76); Human Cervical Cancer Cells (HELA); Canine Kidney Cells (MDCK); Buffalo Rat Liver Cells (BRL 3A); Human Lung Cells (W138) human hepatocytes (Hep G2); mouse mammary tumors (MMT060562); see, eg, 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)), as well as YO , NSO, and Sp2/0 myeloma cell lines. For reviews of specific mammalian host cell lines suitable for antibody production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Assays The anti-integrin β7 antibodies provided herein can be identified or screened for their physical/chemical properties and/or biological activity by various assays known in the art; or can be characterized.

The Etrolizumab Potency Assay measures the ability of Etrolizumab to inhibit RPMI8866 B cell binding to MAdCAM. In this assay, MAdCAM was coated onto 96-well microtiter plates. After overnight incubation, etrolizumab standards, controls and samples were added to the plate along with a fixed amount of cells. Plates were incubated at 37°C in a humidified incubator to allow cells to bind MAdCAM. A washing step is performed to remove non-adherent cells, leaving the remaining viable cells blue and non-fluorescent in the oxidized state, but reduced by the intracellular environment to a highly fluorescent pink color. It was quantified by adding the redox dye Alamar Blue. Therefore, color and fluorescence changes were proportional to the number of bound viable cells. Results were expressed in RFU and plotted against etrolizumab concentration. Parallel curve analysis was used to estimate the activity of etrolizumab sample(s) relative to the reference substance.

Efficacy Assay Articles of Manufacture and Kits An article of manufacture is provided that includes a formulation and provides instructions for its use. Articles of manufacture include containers.

In certain embodiments, articles of manufacture are provided that include a subcutaneous administration device that delivers a dose of an anti-integrin β7 antibody or antigen-binding fragment thereof to a subject, the dose being, for example, but not limited to, 105 mg or 210 mg. not. In certain embodiments, the anti-integrin β7 antibody is etrolizumab. The anti-integrin β7 antibody or antigen-binding fragment thereof in the subcutaneous administration device is formulated in a buffer such as histidine pH 5.8 and other excipients such as polysorbate and arginine succinate to provide a stable pharmaceutical formulation. become.

In certain embodiments, the subcutaneous administration device is a pre-filled syringe comprising a needle and optionally a glass barrel with a needle shield and optionally a needle shield device. In certain embodiments, the volume of formulation contained in the syringe is from about 0.1 mL to about 2 mL, from about 0.1 mL to about 2 mL, from about 0.5 mL to about 2 mL, or from about 1 mL to about 2 mL. In certain embodiments, the volume of formulation contained in the syringe is from about 0.5 mL to about 2 mL. In certain embodiments, the volume of 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 formulation contained in the syringe is about 0.7 mL. In certain embodiments, the volume of formulation contained in the syringe is about 0.75 mL. In certain embodiments, the volume of formulation contained in the syringe is about 1 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is from about 0.5 mL to about 1.0 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is from about 1.0 mL to about 1.5 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.4 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.5 mL. In certain embodiments, the volume of formulation contained in the pre-filled syringe is about 1.45 mL.

In certain embodiments, the needle is a staked-in needle that includes a 3-bevel tip or a 5-bevel tip. In certain embodiments, the needle is 25 gauge (G) to 30G. In certain embodiments, the needles are 1/2 inch to 5/8 inch long. In certain embodiments, the subcutaneous administration device comprises a pre-filled 1.0 mL low tungsten borosilicate glass (Type I) syringe and a stainless steel 5 bevel 27G 1/2 inch long thin-wall stake-in needle including. In certain embodiments, the subcutaneous administration device includes a rigid needle shield. In certain embodiments, the rigid needle shield comprises a low zinc content rubber compound and comprises a rigid polypropylene shield. In certain embodiments, the rubber plunger stopper comprises Daikyo 777-7 rubber and 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, the Ultrasafe Passive® Needle Guard X100L (Becton Dickinson and Company) and Rexam Safe Sound™ (Rexam).

In certain embodiments, the injection device is a pre-filled syringe. Non-limiting examples of pre-filled syringes include BDHYPAK SCF®, READYFILL™, and STERIFILL SCF™ manufactured by Becton Dickinson; CLEARSHOT™ copolymer pre-filled syringes manufactured by Baxter; Daikyo Seiko CRYSTAL ZENITH® pre-filled syringes available from Services. In certain embodiments, the pre-filled syringe comprises silicone oil. Etrolizumab prefilled syringes were developed with optimal levels of silicone oil to ensure low injection forces while keeping the risk of particle formation low. Free fatty acids from degraded polysorbate are split into silicone oil to a greater or lesser extent depending on pH and polysorbate concentration. Therefore, a particular combination of formulation excipients at a combined target pH and target level of silicone oil is uniquely well suited for long-term storage of high-concentration antibody formulations stored in pre-filled syringes. In certain embodiments, the amount of silicone oil in the pre-filled syringe is about 1 mg or less. In certain embodiments, the amount of silicone oil in the pre-filled syringe is from about 0.1 mg to about 1 mg. In certain embodiments, the amount of silicone oil in the pre-filled syringe is from about 0.2 mg to about 0.6 mg. In certain embodiments, the amount of silicone oil in the prefilled syringe is about 0.5 mg to 0.9 mg.

A pre-filled syringe may have any suitable syringe capacity. In certain embodiments, the prefilled syringe has a volume of about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 2.5 mL, about 1 mL to about 5 mL, or about 1 mL to about 2.5 mL. It has a syringe capacity of 5 mL. In certain embodiments, the pre-filled syringe has a syringe capacity of 1 mL. In certain embodiments, the pre-filled syringe has a syringe capacity of 2.25 mL.

In certain embodiments, the injection device is an autoinjector, such as the autoinjector disclosed in US Pat. Nos. 2014/0148763 and 2014/0114247, incorporated herein by reference. In certain embodiments, the autoinjector is a disposable autoinjector. In certain embodiments, the autoinjector is based on Rotaject® technology (e.g., accessible at https://www.shl.group/products-and-services/rotaject-technology-auto-injector/). ing.

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

Kits typically include one or more of the materials desirable from a commercial and user perspective, including containers as described above and buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. and another container of A label may be present on the container to indicate that the composition is used for the particular therapy.

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

Example 1
Important aspects of optimizing etrolizumab drug product formulation and configuration are minimizing viscosity to minimize injection force, maximizing injection volume precision, and optimizing compatibility with device components. Balance optimal formulation excipients to adequately protect antibodies from physical and chemical degradation while maintaining biological activity (potency), achieving high antibody concentrations while maintaining low viscosity It is important to. These aspects of the formulation must be balanced in concert with the pre-filled syringe configuration and specifications (silicone oil concentration, syringe material of construction, barrel width, and needle wall thickness, etc.), resulting in lower It allows for injection force, shorter injection times, and less pain as a result of injection.

In this example, an etrolizumab formulation containing 150 mg/ml etrolizumab at pH 5.8, 20 mM histidine, 200 mM arginine succinate, and 0.04% w/v polysorbate 20 (PS20) was prepared. This etrolizumab formulation was shown to be unexpectedly well suited for long-term storage at high concentrations in pre-filled syringes. Moreover, when paired with pre-filled syringes, the formulation minimizes the risk of sub-visible and visible particle formation during long-term storage, allowing for extended shelf life and patient convenience. The effects of formulation pH, arginine, succinate, polysorbate 20, antibody concentration and histidine on etrolizumab formulation viscosity and stability (eg, aggregate formulations, charge variants, etc.) were investigated.

pH and polysorbate 20
The effects of polysorbate 20 and pH of solutions (formulation buffers) on the solubility of lauric acid, myristic acid and palmitic acid were investigated. As shown in Figure 1, increasing the level of polysorbate 20 increases the solubility of lauric, myristic and palmitic acids (which can be produced as a result of polysorbate degradation), thereby reducing the risk of forming particles. . The results shown in Figure 1 also suggest that increasing pH increases the solubility of free fatty acids. pH 5.8 is the optimal formulation pH to balance the risk of particle formation and antibody chemical and physical stability. This pH formulation minimizes the rate of aspartic acid (Asp) isomerization and succinimide intermediate formation, which can be maintained at ambient temperature without substantially affecting antibody chemical stability and thereby product quality. Allows for patient convenience in combination with the device by allowing storage in . In addition, the increased solubility of free fatty acids at higher pH, which can be attributed to polysorbate degradation, reduces the risk of particle formation.

Arginine Low viscosity is desirable because it can reduce injection force and ensure injection volume accuracy. The use of arginine hydrochloride or arginine succinate in antibody formulations has been previously described. See, for example, US Pat. No. 8,142,776 and International Patent Application Publication Nos. 2006065746 and 2010102241. The effect of arginine succinate on formulation viscosity was investigated. First, the viscosities of etrolizumab formulations containing arginine succinate were compared with those of etrolizumab formulations without arginine succinate, and the results are shown in FIG. As shown in Figure 2, solution viscosity was significantly reduced by the addition of arginine succinate to the formulation. We next investigated whether arginine, in this case 200 mM arginine succinate, could affect the viscosity of etrolizumab formulations at different antibody concentrations. The viscosity of etrolizumab formulations containing 100 mg/mL, 150 mg/mL, 180 mg/mL, 200 mg/mL and 220 mg/mL was measured and the results are shown in FIG. As shown in FIG. 3, with arginine succinate (eg, 200 mM arginine succinate), formulations with antibody concentrations as high as 200 mg/mL have viscosities suitable for pre-filled syringe (PFS) administration.

SILICONE OIL Pre-filled syringe formulations typically contain silicone oil, and because it is known that silicone oil can cause protein aggregation and/or particle formation over time, we have The effect of silicone oil was investigated.

To test the effect of silicone oil, pre-filled syringe glass barrels were sprayed with silicone oil inside and then filled with the etrolizumab formulation. To assess protein aggregation, the high molecular weight species (HMWS) of etrolizumab were determined using size exclusion chromatography (SEC) to determine the aggregation and oligomeric state of antibodies containing HMWS in etrolizumab formulations. 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 different amounts of silicone oil on the formation of HMWS was determined with etrolizumab formulations containing 150 mg/mL etrolizumab or 180 mg/mL etrolizumab. As shown in Figure 4, increasing the level of silicone oil did not affect the physical stability of all etrolizumab formulations tested under various conditions.

Therefore, certain combinations of formulation excipients at target pH and in the presence of silicone oil are uniquely well suited for long-term storage of high-concentration antibody formulations stored in pre-filled syringes. This finding is supported by the long-term stability of etrolizumab in pre-filled syringes for approximately 6 years (eg, 74 months) with no visible particles observed.

Succinate and Histidine The formulations of the present disclosure have the advantage of protecting the antibody from further stress that may result from storage in pre-filled syringes. Leakage from pre-filled syringes can lead to chemical and physical degradation of the antibody. For example, zinc and tungsten can contribute to metal-catalyzed decomposition. During the syringe manufacturing process, a hot tungsten pin is 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 aggregate formation. Tungsten can induce protein aggregation and formation of proteinaceous particles. Protein oxidation can also be induced by tungsten, resulting in protein aggregation. Succinate was protected from zinc-based metal-catalyzed decomposition. Additionally, formulations of the present disclosure were not susceptible to tungsten-spike mediated aggregation.

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

Both the plunger stopper and needle shield may be constructed from a rubber material that allows zinc to leach into the formulation. It is known that zinc can complex with proteins, resulting in protein aggregates (eg HMWS) and increased viscosity of antibody formulations. The effect of zinc on the viscosity of etrolizumab formulations at various protein concentrations was investigated. As shown in Figure 6, in formulations containing 20 mM histidine, 200 mM arginine succinate at pH 5.8, when the concentration of etrolizumab was 150 mg/mL or less, there was a difference between zinc-free and 10 mM zinc-containing formulations. There was no difference in viscosity. Zinc increased viscosity at etrolizumab concentrations above 150 mg/mL. Further, Figure 7 shows protein aggregate formation in formulations containing 150 mg/mL etrolizumab and 50 mM zinc. FIG. 8 shows that addition of zinc increases the percentage of HMWS, and the increase in percentage of HMWS is higher with 50 mg/mL etrolizumab compared to 10 mg/mL etrolizumab.

We then tested whether histidine could reduce the formation of HMWS. As shown in Figure 9, the presence of histidine in the formulation significantly reduced the rate of HMWS formation in the presence of zinc. Furthermore, as shown in Figure 10, succinate also inhibited the interaction between etrolizumab and zinc to form HMWS. Next, the effect of varying concentrations of histidine and succinate on HMWS formation was studied. As shown in Figure 11, the combination of histidine and succinate minimized HMWS formation in the presence of zinc.

Drug Substance and Drug Substance Stability Etrolizumab drug substance (DS) formulated at 150 mg/mL in 20 mM histidine, pH 5.8, 200 mM arginine succinate, 0.04% polysorbate 20 was tested for stability at and -20°C. Stability was also evaluated 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 storage of DS at -20°C for 7 years (84 months). After 6 months of storage at 5°C no changes were observed in all assays except ion exchange chromatography. After storage for 14 days at 30°C, degradation was determined by non-reducing capillary electrophoresis, ion exchange and size exclusion chromatography.

Deamidation and isomerization are possible degradation pathways for stability. Chemical degradation was monitored using ion exchange chromatography (IEC) to quantify acidic variants, basic variants, and the main peak. No changes were observed in the main peak, acidic or basic variants after 5 freeze/thaw cycles or after storage at -20°C for 84 months. After storage at 5° C. for 6 months, a loss of 1.7% of the main peak accompanied by 1.0% and 0.8% was observed in the acidic and basic variants, respectively. After storage for 14 days at 30° C. there was a 4.5% loss of the main peak, accompanied by a 2.1% and 2.4% increase in acidic and basic variants, respectively. Qualitatively, no change in ion exchange profile or new peaks were observed.

After storage for 14 days at 30° C., the following changes were observed: By non-reducing capillary electrophoresis—SDS, a loss of 1.0% of the main peak was observed, concomitant with a pre- and post-peak of 0.00 each. 82% and 0.14% increases; by IEC there was a 4.5% loss of the main peak, accompanied by a 2.1% and 2.4% increase in the acidic and basic variants, respectively; By size exclusion chromatography (SEC), 28% and . An increase of 09% is observed.

The stability of etrolizumab drug product 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 configuration was investigated. Etrolizumab showed no change in product quality after 60 months at 5°C as assessed by clarity, opacity and color (COC), pH, protein concentration, size exclusion chromatography (SEC) and potency. A 7.7% loss of the main peak by ion-exchange chromatography (IEC) with a concomitant increase in acidic and basic variants of 4.2% and 3.4%, respectively, and a main peak by non-reducing CE-SDS. There was a 1.0% loss of peak. No change in clarity, opacity and color (COC), pH, protein concentration or potency after storage at 25°C for 3 months, size exclusion increasing by 0.2% for both high and low molecular weight species There is a 0.4% loss of the main peak by chromatography, a 1.3% loss of the main peak by CE-SDS, a 5.1% gain for acidic variants and a 4.6% gain for basic variants. There was a 9.8% loss of the main peak by ion exchange chromatography with a % increase. Etrolizumab drug product stored at 5° C. for 60 months was substantially free of visible particles.

CONCLUSIONS Unique aspects of certain excipient combinations in etrolizumab formulations can protect the antibody from additional stresses that can result from storage of the antibody in pre-filled syringes, e.g. HMWS formation by antibodies can be reduced. A typical antibody formulation contains 0.02% w/v polysorbate 20 and a typical antibody formulation has a pH of 5.5. Higher than typical polysorbate 20 concentrations (e.g., 0.04% w/v) and higher than typical pHs (e.g., pH 5.8) can solubilize free fatty acids, thereby , reducing the risk of particle formation as a result of polysorbate degradation during long-term storage, for example in pre-filled syringes. The combination of excipients in etrolizumab formulations is particularly effective in controlling pH during tangential flow filtration (TFF) in large scale manufacturing. High arginine concentrations and high conductivity formulations shield the charge on the antibody and prevent pH shifts. The histidine concentration effectively buffers the formulation at the target pH. This is important to ensure robust control of pH during manufacturing and to allow a narrow pH range of the formulation to allow for this optimal formulation. The formulations also maintained chemical and physical stability and maintained potency at various temperatures for various periods of time, as described above.

Example 2 - Clinical Strategy and Initial Outcomes in Supporting an Etrolizumab Autoinjector in Healthy Volunteers Summary It is an anti-β7 integrin antibody. The Phase 3 trial will utilize a pre-filled syringe (PFS) for etrolizumab administration. In parallel, automatic injection devices (AI) are being developed to increase patient delivery options. This example describes the overall development strategy and details the first-in-human research of this development AI.

This open-label study in healthy volunteers (HV) evaluated the tolerability and efficacy of an investigational etrolizumab AI. The primary endpoint was the proportion of participants with more than mild pain after injection. Adverse events (AEs) and use errors were also evaluated. Results were reported by injection site (thigh vs. abdomen) and needle training (experienced vs. naive). Pharmacokinetic (PK) variability between participants was included as an exploratory endpoint.

Thirty participants completed the study. A total of 97% of participants never experienced more than mild pain during the study, and 50% experienced no pain. Three use errors were observed, one of which resulted in partial dose delivery of etrolizumab. No patterns of usage errors were observed. Mild injection site reactions (ISR) were reported and all resolved by the end of the study. Participants with abdominal injections reported more ISRs than those with thigh injections, and needle training did not appear to affect the incidence or severity of AEs.

Results from this first-in-human study demonstrate that a single injection of etrolizumab 105 mg using an AI is well tolerated in HV with transient mild pain and minimal error of use. . The results of this study also informed the design of subsequent PK equivalence studies comparing PFS and AI. Overall, the availability of AI may offer an attractive option for patients desiring a convenient and easy-to-use delivery mechanism for etrolizumab.

Introduction Etrolizumab has been evaluated in an extensive clinical program of Phase 3 trials in patients with moderate-to-severe UC and Crohn's disease (Etro Studies. The Etro Studies: Explore Innovation: Contribute to Science. Genentech; 2019.2019). Accessed July 26, 2009), etrolizumab is administered once monthly by subcutaneous (SC) injection using a prefilled syringe (PFS) with a needle safety device (NSD).

Single-use pre-filled autoinjectors (AIs) have many potential advantages over PFS-NSDs, most notably the ability to keep the needle out of the user's sight at all times during injection. be. Exemplary PFS and AI are shown in FIG. AI also offers increased convenience, ease of use, reduced risk of dose errors, and increased patient comfort. Studies consistently show that many self-administering patients prefer AI to 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. AL. 12:1193-202). . For example, a recent study of golimumab in UC patients demonstrated that the majority of patients preferred dosing with an AI compared to PFS, citing increased ease of use and reduced injection discomfort ( Vermeire et al., Patient Prefer Adherence.2018;12:1193-202).

The AI currently in development consists of an automated delivery system housing the same PFS used in the Phase 3 trial (see Figure 13). The formulation included in both AI and PFS-NSD consisted of a 105 mg liquid formulation of etrolizumab solution (0.7 mL, nominal volume 150 mg/mL) for single dose administration. The entire dose is typically dispensed in about 2 seconds.

AI includes a number of features aimed at improving patient experience and enhancing patient comfort with self-administration. Automated drug delivery systems are activated by gently pressing the device vertically onto the skin. When activated, the AI automatically inserts the needle and dispenses the syringe contents upon activation. Once the injection is complete, a needle cover extends over and locks the needle, keeping the needle out of sight at all times during injection and protecting the user and others from accidental contact with the used needle. The AI also incorporates both visual and auditory mechanisms designed to assist the user with self-injection, with both a visible spin-top and an audible click indicating whether drug administration is in progress. Indicates whether it is complete. Additionally, the visible plunger rod moves across the viewing window as the injection progresses.

Here we present an overall strategy for developing a novel AI for etrolizumab and findings from first-in-human studies of this device. The primary objective of this study (NCT02629744) was to assess the safety and tolerability of etrolizumab administered by an AI and primarily injection site pain following self-injection by an AI and document significant use errors. Met. In addition, we evaluated this relatively unique study design, which combines use error assessment (traditionally done as a simulated study) with tolerability, safety and exploratory PK studies. It was something.

Methods Research plan and procedures. This first-in-human AI tolerability study was an open-label, single-arm study in healthy volunteers evaluating the pain, safety, and efficacy of AIs when self-administered subcutaneously. Participants were assigned to two groups (1:1). To simulate previous experience of self-injection, one group (“Needle Experienced”) was trained prior to self-injection by the AI and the other group (“Needle Naive”) was not trained. Ta. Training included a simulated needle experience by self-injection with a placebo. Prior to etrolizumab administration, all participants (regardless of needle experience group) received an Instructions for Use (IFU) leaflet on the AI for review prior to self-injection. Subjects were randomly assigned to receive study drug in the abdomen or anterior thigh. All participants self-administered a single SC dose of etrolizumab on study day 1 in a simulated home setting (see Figure 14). Participants were monitored during self-injection, discharged on study day 3, and attended for follow-up visits on study days 8, 29, 43, 57, and 85 (study completion).

participant. Eligible participants were between 18 and 65 years of age, had a body mass index (BMI) between 18.0 and 32.0 kg/m ² or less, and were in good health with no significant medical history or laboratory abnormalities. Both males and females were enrolled with the goal of 55%-60% male participants to mimic the gender distribution of patients with IBD. Participants with any previous use of anti-integrin therapy (including etrolizumab) or immunosuppressive drugs were excluded, as were participants with recent history of corticosteroid use. Participants with a history of tuberculosis were also excluded.

procedure. Participants assigned to the needle experienced group underwent training (a simulated self-injection experience) 5 and 7 days prior to etrolizumab injection. During training, participants were instructed by a medical professional on the use of needles and syringes. Following this instruction, participants self-injected 3 times with placebo solution using a needle and syringe. Needle-experienced participants deemed suitable by a medical professional (based on interaction with the syringe) progressed through the study. Both needle-experienced and needle-naive participants were randomized, stratified by gender and needle experience, and injected into either the abdomen or the anterior thigh. On Study Day 1, participants self-administered a single SC dose of 105 mg etrolizumab into their abdomen or anterior thigh using an AI. Participants were rated for operational difficulties and errors in use and reported pain during and immediately after injection. One serum sample was collected for exploratory pharmacokinetic evaluation 7 days after SC injection (study day 8).

Approaches for assessing pain. Pain was assessed by two independent methods, both of which were administered by study site personnel. The 7-point Categorical Verbal Descriptive Scale (VDS-7) was the primary measure of pain in this study. During VDS-7 administration, patients were asked to select a number from 1 to 7 that best describes the pain associated with the injection (on a scale as follows: 1 = no pain, 2 = very mild pain). , 3 = mild pain, 4 = less severe pain, 5 = very severe pain, 6 = very severe pain, 7 = barely intolerable pain). As a confirmatory assessment, pain was also assessed by a 100-point continuous visual analogue scale (VAS). For the VAS, patients were asked to draw a horizontal 100 mm scale line that best represented their pain (scale as follows: 0 mm = no pain, 100 mm = worst possible pain). Investigators then measured the distance between the 0 mm point and the patient's mark to determine the patient's VAS score.

Outcome. The primary endpoint was the proportion of participants with greater than mild pain (VDS-7>3) immediately after injection. To meet the primary endpoint, the upper limit of the two-sided 95% confidence interval (CI) centered on the proportion of participants experiencing more than mild pain immediately after injection should not exceed 30%. Secondary endpoints were the proportion of participants experiencing greater than mild pain at 5, 10, 20, 60, and 240 minutes (4 hours) after injection and participation in each VDS-7 category over time. including the percentage of Tolerability was assessed in this study by active monitoring for injection site reactions (ISR) at 5, 60, and 240 minutes post-injection on Study Day 1 and on Study Days 2, 8, 43, and 85. evaluated intensively. To identify ISR, a Local Injection Site Symptom Assessment (LISSA) was performed to assess the formation of burning, itching, bruising, redness, and/or urticaria, and the magnitude of reaction, if present. All ISRs were classified and reported as adverse events (AEs) or serious AEs as appropriate. Documented usage errors and operational difficulties while using the AI. In addition, participants' IFU knowledge and overall opinions about their AI experience were collected. Safety was assessed by AE monitoring, laboratory assessment, vital signs, physical examination, electrocardiogram (ECG), and immunogenicity. No formal statistical study was planned for this study. Determination of PK fluctuations at a single time point of the study on day 8 after self-injection was evaluated as the exploratory endpoint.

Results Thirty healthy participants were enrolled and randomized (stratified by gender and needle experience) to receive etrolizumab injections in the abdomen or anterior thigh. Although all participants completed the study, one volunteer (needle-experienced femoral injection) did not receive the full dose of etrolizumab due to usage error (discussed further). The enrolled population was broadly representative of the IBD population. The median age of enrolled participants was 36 years, the mean BMI was 26.1 kg/ ^m2 , and the majority of participants were Caucasian (60%), Panic or Latino (83%). ) was not. About half of the participants (47%) were male.

Pain and Tolerability. Half of the participants reported no pain at any time after injection (see Figure 15). Of the participants who reported pain, all but one reported "very mild" or "mild" pain and most subsided within 60 minutes after 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 were reported using VAS (data not shown). Reported pain varied between injection sites. Injections in the thigh increased the proportion of participants reporting pain compared to participants injecting in the abdomen (60% vs. 40%, respectively) (see Figure 16). One volunteer who reported more than mild pain was assigned to the thigh dose group. Participants who inject into the thigh also reported pain of longer duration than those who injected into the abdomen, with all pain experienced after abdominal injection resolving within 5 minutes and less than 5 minutes after thigh injection. Most of the pain subsided within 60 minutes. Needle experience training did not appear to affect reported pain after etrolizumab injection. Using the focused LISSA-based monitoring scheme described above, 40% (12/30) of participants experienced ISR during the study, all of which occurred within 1 hour after etrolizumab injection (Table 1).

All reported ISRs were mild (Grade 1) and transient, and all resolved by study completion. The most frequent ISR was redness and ranged from less than 18 mm to 31 mm in diameter. Most ISR resolved within 60 minutes after injection. One volunteer reported the formation of urticaria (18 mm in diameter) at the abdominal injection site 60 minutes after dosing, and the urticaria resolved within 3 hours without treatment. Injection site did not appear to affect either the frequency or severity of ISR.

Usage error. Twenty-seven of the 30 participants (90%) were able to successfully self-administer etrolizumab using the AI without significant error in use, regardless of needle experience training. No AI complaints were recorded and no patterns of usage errors were observed. A total of three use errors were observed during the study, only one of which occurred during infusion. One volunteer began removing the AI prematurely during the injection, resulting in droplets remaining on the volunteer's skin. Of the two use errors that did not occur during injection, one volunteer was unsure when to remove the cap from the AI and another incorrectly reported the simulated expiration date. These two usage errors were related to AI labeling and IFU misinterpretation, and neither of these errors affected the dose of etrolizumab administered. Most participants said they found the AI easy to use. Participants reported that the audible and visual feedback mechanisms were useful in determining when injections started and stopped and verifying that the full dose was administered. However, some participants stated that it was difficult to see the spintop visually while injecting into the abdomen. During the IFU comprehension probe, some confusion was noted regarding acceptable injection sites, drug warm-up time, and product storage.

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

safety. Twenty-nine participants (97%) received the full 105 mg dose of etrolizumab. One volunteer received approximately 90% of the 105 mg dose. Overall, a single 105 mg SC dose of etrolizumab was safe and well tolerated when self-administered using an AI. Approximately half of the participants experienced treatment-emergent adverse events (TEAEs), most of which were injection site related. All TEAEs were mild in severity (Grade 1) and resolved by study completion. No significant changes in clinical laboratory assessments, vital sign measurements, body weight measurements, or 12-lead ECG were observed during this study. Interestingly, more TEAEs were reported by participants injected in the abdomen compared to the thigh (19 vs. 9 TEAEs, respectively). Fewer TEAEs were reported by needle-experienced participants than by needle-naive participants. A summary of all results can be found in Table 2.

Discussion In healthy participants, a single self-administered SC dose of etrolizumab with AI was well tolerated and produced mild pain in the majority of participants. This study met its primary endpoint of only one volunteer experiencing more than mild pain after injection. Overall, the data presented here are consistent with those of AIs used to treat other chronic diseases, including rheumatoid arthritis (RA) and chronic kidney disease. These studies suggest that many patients prefer the convenience of AI compared to PFS. Patients generally report that AI is associated with less pain than PFS devices and that they perceive AI as more portable and easy to use (Kivitz et al., Clin Ther. 2006;28(10):1619). 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 patients with UC reported similar findings, noting that approximately three-quarters of patients in this study preferred AI injections compared to PFS (Vermeire et al., Patient Prefer Adherence. 2018; 12:1193-202). A recent multinational survey asked 200 patients with RA and 100 nurses to rate the relative importance of various components of AI (Tischer and Mehl, Patient Prefer Adherence. 2018; 12 : 1413-24). Both patients and nurses rated "ease of self-injection with pen (ie, autoinjector)" as the most important attribute. Other important attributes reported by both patients and nurses included "needle safely concealed within syringe body,""audible feedback after completion of injection," and "visual feedback after completion of injection." feedback” and all of these features are incorporated into Etrolizumab AI. Similar results were reported in a European study of 220 patients with RA (Thakur et al., Rheumatol Ther. 2016;3(2):245-56). It is noteworthy that the proportion of patients experiencing mild ISR in this study was higher than that observed in the Phase 2 EUCALYPTUS study, in which etrolizumab was administered by vial and syringe (Vermeire et al., Lancet .2014;384(9940):309-18). This study focused on assessing tolerability using LISSA to actively monitor ISR at scheduled intervals, which in some cases resulted in over-reporting of ISR. It was thought that there is a high possibility that it reflects the difference in This first-in-human study is relatively unique in that it combines a tolerability assessment, a human factor assessment of actual use (such as error in use), and an initial PK assessment into a single study. This novel approach will, in part, help assess the overall risks associated with AI in a manner that minimizes the number of clinical studies required, thus reducing the overall time and cost of AI development. It is an object. PK assessment was incorporated into the study protocol on study day 8, using a single blood sample drawn around the time of maximum serum concentration following a single SC dose. The intent of this exploratory PK assessment was to understand inter-subject exposure variability following etrolizumab SC delivery by AI. In addition, these preliminary PK data were used to compare potential differences in exposure after AI injection with predicted exposure using a model generated based on PK data from vial and syringe administration in UC patients. helpful in evaluating. Of note, the observed day 8 etrolizumab exposure in this analysis was approximately 75% higher than predicted (predicted day 8 median etrolizumab serum concentration ≈ 7.9 μg/mL [90 % CI 4.15-16.3], data not shown). The PK variability from this analysis and the unexpectedly higher Day 8 exposure led to the decision to conduct a two-part study comparing the pharmacokinetics of etrolizumab delivered by AI and PFS-NSD in healthy volunteers ( See Figure 17 and Example 3). The results from this study effectively eliminated the need for further AI usability and/or clinical studies. Furthermore, these results will influence the design of subsequent studies to compare PK profiles between administration with PFS-NSD and AI, reduce the risk of failure in equivalence studies, and reduce the risk of failure to biologic therapy. Minimize unnecessary exposure of healthy volunteers. As a result of the PK findings reported here, we added a pilot cohort to the originally proposed single-part device PK equivalence study plan. Results from this pilot cohort helped optimize the design of the pivotal cohort by informing appropriate sample size, sampling period, and weight range. Information obtained from the human factor component of this study resulted in minor modifications of the IFU performed prior to the PK equivalence study. Device PK equivalence results are reported in Example 3.

Conclusions The results of this study demonstrate that a single SC injection of etrolizumab with AI is well tolerated in healthy volunteers and is associated with acceptable levels of pain following injection. Most participants found the AI easy to use and experienced minimal use errors. AIs may be an appropriate delivery mechanism for certain patients with IBD who desire the safety and convenience of self-injection through an invisible needle. Positive results from this first-in-human tolerability study, combined with data collected during a subsequent two-part PK equivalence study, will support the use of AI in patients treated with etrolizumab for full development Including plans.

Example 3 - Comparable Pharmacokinetics, Safety and Tolerability Summary of Etrolizumab Administered via Prefilled Syringe or Autoinjector in a Randomized Study in Healthy Volunteers is a novel dual-acting anti-β7 integrin antibody being investigated in several phase 3 trials in . A parallel autoinjector (AI) device is being developed to complement the pre-filled syringe (PFS) with the needle safety device (NSD) used for subcutaneous administration in these trials. This example demonstrates comparability of pharmacokinetics (PK), tolerability and safety of both devices.

This randomized, open-label, two-part study in healthy participants evaluated the equivalence of etrolizumab exposure between AI and PFS-NSD. Part 1 (pilot) involved a small number of participants and initial results were used to complete a larger Part 2 (pivotal) study plan. In both parts, participants were randomized to receive a single dose of subcutaneous (SC) etrolizumab 105 mg via AI or PFS-NSD. Randomization was stratified by weight. Primary PK outcomes were C _max , AUC _last , and AUC _0-inf .

180 healthy participants (Part 1: n=30, Part 2: n=150) received a single SC dose of etrolizumab via AI or PFS-NSD. Primary PK results from Part 1 supported modifications to the Part 2 study plan. Results from Part 2 showed that etrolizumab exposure was comparable between devices, with geometric mean ratios (GMR) between AI and PFS-NSD of 102% for C _max , 98.0% for AUC _last and AUC _0-inf was demonstrated to be 97.6%. Median T _max and mean terminal t _1/2 were also similar between devices. The GMRs and 90% confidence intervals of all primary PK parameters were completely within the prescribed equivalence limits (80%-125%).

This PK study demonstrated that a single SC injection of 105 mg etrolizumab using AI or PFS-NSD resulted in comparable etrolizumab exposure and similar safety and tolerability in healthy participants. Taken together, these results support the use of AIs for etrolizumab administration.

Introduction The PK equivalence study (NCT02996019) presented in this example demonstrated equivalence of etrolizumab exposure after SC administration using AI and PFS-NSD, and etrolizumab following SC injection using two devices. The aim was to evaluate the safety and tolerability of Part 1 of the study was an exploratory pilot cohort used to assess the variability of geometric mean ratio (GMR) and PK parameters of etrolizumab administration with AI versus PFS-NSD. These results informed the study plan, including sample size and study period for Part 2 (pivotal cohort). In Part 2, the study aimed to demonstrate exposure equivalence between single-dose etrolizumab administered via AI or PFS-NSD. The PK equivalence study presented in this example leveraged exploratory PK results from the tolerability study presented in Example 2 to refine the study design and final protocol.

Methods Research plan and procedures. This study was a randomized, multicenter, open-label, parallel group study conducted in healthy participants at three clinical centers in the United States (see Figure 18). This two-part study consisted of a pilot cohort (part 1) and a pivotal cohort (part 2) with sample sizes sufficient for 80% power to detect exposure differences (if any) between the two device groups. . In both parts, healthy participants were randomized 1:1 to receive a single dose of etrolizumab 105 mg SC via either AI (test device) or PFS-NSD (reference device). Etrolizumab was administered to the participant's abdomen by a health care professional (HCP). Randomization in both cohorts was stratified by body weight (≤79.9 kg vs. ≥80 kg).

participant. Eligible healthy participants included men and women aged 18-55 with a body mass index (BMI) of 18.0-30.0 kg/m ² . Based on results from the pilot cohort, participants in the pivotal cohort were also required to have a weight within the range of 60-100 kg (inclusive) at study entry. Participants must be in good health (no clinically significant findings from medical history, physical examination, 12-lead ECG or vital signs). Participants with previous exposure to immunosuppressive agents or anti-integrin therapy (including etrolizumab) were excluded.

Evaluation and Measurement of Results Blood samples for determination of both Part 1 and Part 2 etrolizumab serum concentrations were administered predose and 6 hours postdose on Day 1, then 2, 4, 6, 8, 11, 15, 2, 4, 6, 8, 11, 15; Harvested on days 29, 43, 57, and 71 (study completion). Geometric mean ratio (GMR) and variability of maximum etrolizumab concentration (C _max ), area under the concentration-time curve from time of drug administration to last measurable concentration (AUC _last ), AUC extrapolated to infinity (AUC _0-inf ), and the ratio of AUC _last to AUC _0-inf of etrolizumab (AUCR) were determined in Part 1. For Part 2, C _max , AUC _last , and AUC _0-inf were measured as primary endpoints. Secondary PK parameters included time to maximum concentration of etrolizumab (t _max ), terminal elimination half-life (t _1/2 ) and AUCR. Blood samples for determination of anti-drug antibodies (ADA) for Parts 1 and 2 were collected on Day 1 and on Days 29, 57, and 71 prior to dosing. Data from all ADA-positive participants were included in the final PK statistical analysis unless the participant met predetermined exclusion criteria for PK analysis. Evaluation of safety and tolerability 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. included severity. Incidence of injection site reactions, changes in vital signs, physical examination findings, laboratory test results, and incidence of ADA were also assessed.

bioanalytical method. Etrolizumab concentrations were measured using a validated immunoassay (ICON). This Gyrolab fluorescence immunoassay utilized a minimum required dilution (MRD) of 1/100 with a minimum quantifiable concentration of 80 ng/mL in UC, CD and healthy volunteer serum. Anti-etrolizumab antibodies in serum were detected using a validated assay. This colorimetric ELISA used a 1/20 MRD and a monoclonal anti-etrolizumab control antibody. The relative sensitivity of this method was determined to be 12.0 ng/mL in healthy volunteer serum. The drug tolerance of the assay was established: 28 ng/mL positive control ADA could be detected in the presence of 50 μg/mL etrolizumab.

Determination of sample size. With a plan to enroll a maximum of 30 healthy participants in Part 1, at least 12 participants in each arm (24 total) had an evaluable PK profile and PK parameters ( _Cmax , It was confirmed that the GMR and coefficient of variation (CV%) for AUC _last and AUC _0-inf ) could be estimated. Part 2 planned to enroll 146 participants. A total of approximately 131 healthy participants with evaluable full PK profiles, assuming a dropout rate of approximately 10% of participants by Day 71 or a non-evaluable PK profile, were GMR from the Part 1 pilot cohort. and expected to provide at least 80% power to demonstrate exposure equivalence for C _max , AUC _last , and AUC _0-inf based on PK variability outcomes.

Pharmacokinetic analysis. PK parameters were determined from serum etrolizumab concentrations using non-compartmental methods (NCA) and performed using Phoenix WinNolin (Centara USA, Inc., version 6.4).

statistical analysis. The analysis population consisted of all participants who received SC injections of etrolizumab and had an evaluable PK profile, defined as having sufficient sample available to accurately determine key PK parameters. Specifically, participants who terminated early before Day 15 were considered not to have a measurable PK profile. Participants with no samples available to determine concentrations on Day 71 were excluded from the statistical analysis of AUC _last , and participants with less than 3 samples available on Days 28, 43, 51, and 71 were excluded from the statistical analysis of AUC _0-inf . Participants with pre-dose concentrations greater than 5% of C _max may be excluded from the statistical analysis of all PK parameters at the discretion of the study and the sponsor's clinical pharmacokinetic/biostatistician. Using data from Part 1 (pilot), a descriptive, exploratory analysis of PK parameters was performed, with a focus on assessing the distribution of GMR, CV% and AUCR, sample size and final I informed him of my research plan. Only data from the pivotal cohort (Part 2) were included in the equivalence statistical analysis. In Part 2, ANOVA with treatment as a fixed effect was performed to assess the equivalence of C _max , AUC _last , and AUC _0-inf between the AI and PFS-NSD groups. By natural log (ln) transforming the C _max , AUC _last , and AUC _0-inf data before analysis and taking the antilog of the corresponding 90% confidence interval (CI) for the difference between the means (log scale) , the 90% CI of the GMR of the AI group relative to that from the PF-NSD group was calculated. Exposures between AI and PFS-NSD groups met PK equivalence criteria if the 90% CIs of GMR for C _max , AUC _last , and AUC _0-inf were all within 80%-125% .

Results Pharmacokinetics - pilot study (part 1). All 30 participants were enrolled and randomized in Part 1 to receive a single 105 mg SC dose of etrolizumab via AI (n=15) or PFS-NSD (n=15). Twenty-seven participants completed the study, 2 participants (1 in each group) discontinued prematurely due to loss of follow-up, and 1 experienced a serious AE 32 days after administration of etrolizumab ( seizures) (PFS-NSD group) and this AE was not considered related to etrolizumab treatment. Table 3 shows the characteristics of the participants.

Across the cohort, most participants were male (63.3%) and Caucasian (70.0%). The weight and age of the participants were not balanced between treatment groups, with a mean weight of 79.7 kg in the AI arm and 74.3 kg in the PFS-NSD arm, and a mean age of 42 years in the AI arm and PFS-NSD arm. He was 34 years old with an NSD arm. Since body weight appears to influence etrolizumab exposure, particularly AUC _0-inf (see Figure 19), imbalance in body weight between treatment arms biased the assessment of the primary PK parameters GMR values and PK variability. There is a possibility that Therefore, participants in the pilot cohort with a body weight of less than 60 kg (n=4) or more than 100 kg (n=1) were significantly affected by the PK parameters used to guide the determination of the final sample size of the pivotal study (Part 2). Excluded from exploratory analyzes aimed at assessing GMR and CV%. The PK evaluable population meeting the 60-100 kg weight limit in Part 1 included 14 participants in the AI arm and 11 participants in the PFS-NSD arm. As expected, when the weight range between the two arms was balanced by restricting body weight to the range of 60-100 kg, the primary PK parameter GMR values for the AI vs. PFS-NSD group were C _max , AUC _last , and AUC _{0 -inf} were 0.95, 1.02 and 1.02, respectively (Table 4).

Therefore, we calculated the sample size of the pivotal cohort based on these GMR and CV % values and added weight restriction as an inclusion criterion for the Part 2 study. In addition, all participants had AUCR values greater than 80% (data not shown), which is in line with US Food and Drug Administration (FDA) guidelines (US Department of Health and Human Services. Industry Guidance: Bioequivalence). Accessed March 4, 2020) and therefore met the requirements for a bioequivalence study described in the originally planned Day 85 to Day 71 Resulted in a change in the final date of the pivotal study.

Pharmacokinetics-Pivotal Study (Part 2). In the pivotal cohort, 150 (100%) of enrolled and randomized participants received a single SC dose of 105 mg etrolizumab via AI (n=74) or PFS-NSD (n=76). received. 8 participants discontinued the study, 5 lost to follow-up (3 in AI arm, 2 in PFS-NSD arm), 1 protocol violation not meeting weight criteria (PFS-NSD), 2 There were participant withdrawals (one in each arm). In overall Part 2 (both arms included), 53.5% of participants were male, 60.7% were Caucasian, and 36.0% were African American. Treatment groups were balanced for weight and age, with a mean weight of 76.2 kg in the AI arm and 76.3 kg in the PFS-NSD arm, and a mean age of 35 years in the AI arm and 37 years in the PFS-NSD arm. was (Table 3).

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

The 90% CI of GMR for each of these primary PK parameters was within the pre-determined equivalence limits of 80% to 125%, which met the pre-determined equivalence criteria and resulted in comparable exposure of etrolizumab between the two device groups. support. Results also showed similar median time to maximum observed concentration (t _max , 5.04 vs. 6.97 days) and mean terminal elimination half-life (t _1/2 ) between the AI and PFS-NSD groups. , 11.8 vs. 12.2 days).

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

One participant in the PFS-NSD arm of the pilot cohort had a severe AE (grade 3 seizure) approximately 32 days after injection, leading to study discontinuation and not considered related to treatment with etrolizumab. I didn't. Nine participants in the pilot cohort (5 with AI and 4 with PFS-NSD) and 1 participant in the pivotal cohort (AI arm) had an injection site reaction reported as a TEAE. Ta. Treatment-related TEAEs in the pilot cohort were experienced by 6 (40.0%) participants in the AI arm and 3 (20.0%) in the PFS-NSD arm, and 7 (9) in the AI arm in the pivotal cohort. .5%) and 10 (13.2%) in the PFS-NSD arm.

Discussion The pivotal cohort of this study confirmed comparable etrolizumab exposure between the AI and PFS-NSD groups after a single dose of SC etrolizumab in healthy participants. The observed GMR for each primary PK parameter ranged from 98% to 102% with 90% CIs within the prescribed equivalence limits. These GMRs for all primary exposure parameters are impressive given 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 adalimumab biosimilar (SB5) showed GMRs of 102%, 107% and 110% for _Cmax , _AUClast and _AUCinf , respectively, The 90% CI was within the equivalence limits of 80%-125%. In a similar study of adalimumab biosimilar BI695501, they were 100% for AUC _0-inf (90% CI 82.1-122.3%) and 110% for C _max (90% CI 96.8-125.4). %), the latter surpassing the 90% CI equivalence limit (Voltaire®-AI study) of 125% (Ramael et al., Rheumatol Ther. 2018;5(2):403-21; Shin et al., Drug Des Devel Ther. 2018;12:3799-805). A pilot cohort was informative to inform the final design of the pivotal cohort. The pilot cohort primarily assessed variability in GMR and PK parameters, which are key assumptions used in sample size estimation. Intended to obtain certainty around GMR values and PK variability of key PK parameters after administration of etrolizumab by AI or PFS-NSD to minimize the risk of underpowering pivotal PK equivalence studies , added a small pilot cohort to the original study protocol. GMR and PK variability values obtained from the pilot cohort (see Table 4) provided increased confidence in estimating sample size for the pivotal cohort. Although weight was stratified at randomization, the final weight distribution range was still disproportionate in the pilot cohort, which may have biased the final GMR results. To minimize such bias, only data from participants weighing within the 60-100 kg range (a weight range common to both groups within the pilot cohort) were used for estimating GMR and PK variability. did. This weight limit of 60-100 kg was also implemented in the pivotal cohort as an inclusion criterion. The pilot cohort also evaluated a 10-week (70-day) study period, two weeks shorter than the planned study period of the original study suggested by the FDA. As expected, all evaluable participants in the pilot cohort had AUCR values greater than 80%, the value required by the FDA for PK equivalence studies. This result indicates that the 10-week study period was long enough to capture more than 80% of the AUC _inf , thus the period of the pivotal study was extrapolated to less than 20% for the calculated AUC _inf . suggested that 12 weeks could be shortened to 10 weeks without the risk of missing the AUC requirement. This study was relatively immunogenic compared with other studies using etrolizumab. The rate of ADA was >20% in both the pilot and pivotal cohorts, but single or multiple Five percent (2/38) of patients with UC who received etrolizumab dosing and multiple doses of etrolizumab in a randomized phase 2 trial (Vermeire et al., Lancet. 2014;384(9940):309-18) Five percent (4/81) of patients with UC who received had detectable ADA after etrolizumab treatment. The incidence of immunogenicity in the pilot and pivotal cohorts was comparable in both the PFS-NSD and AI groups. The higher ADA rates in this study may have been the result of differences in study design, and the study had a single SC dose regimen and a study population of healthy participants not taking immunosuppressive drugs. Was. In contrast, patients in previous trials of etrolizumab had poorly controlled, moderate to severe UC and were generally treated with immunosuppressants. Although the incidence of ADA after a single SC injection of etrolizumab was relatively high (>20%) in this study, the PK profile of ADA-positive participants largely overlapped with that observed in ADA-negative participants. Given that, the effect of ADA positivity on PK appears to be minimal (Fig. 21). Furthermore, the variability (CV%) of AUC _0-inf or AUC _last values ranged from 33% to 36% in the pivotal 2 study, consistent with that observed for other monoclonal antibodies (Ramael et al. al., Rheumatol Ther. 2018;5(2):403-21; Anumolu et al., Clin Pharmacol Drug Dev.2018;7(8):829-36). These small exposure variations further confirm that the total exposures (AUC _0-inf ) between ADA-positive and ADA-negative participants were very similar. Data from all evaluable participants, regardless of ADA status, were included in the equivalence statistical assessment, and the final results showed a high degree of exposure similarity between groups. Bioequivalence testing and sample sizing of the AI and PFS-NSD groups in the pivotal study design after a single SC dose of etrolizumab was based on data from a subset of participants within the 60-100 kg weight range. Our findings of comparable etrolizumab exposure between AI and PFS-NSD were based on data from participants within a defined weight range, although this study did not demonstrate differences in exposure due to the drug delivery device alone. can be applied to individuals outside this weight range, given that we did not specify Moreover, similar studies with another therapeutic antibody (not weight-limited) suggest that this may be a valid assumption (Ramael et al., Rheumatol Ther. 2018; 5(2) : 403-21). Although similar relationships between drug exposure and body weight were observed in that study, bioequivalence was still achieved regardless of body weight when comparing the AI and PFS-NSD devices for adalimumab SC administration. was done. Clearance of etrolizumab is known to be significantly affected by body weight ((Tang et al., Aliment Pharmacol Ther. 2018;47(11):1440-52), findings confirmed in the pilot cohort of this study. In these healthy participants, single SC doses of etrolizumab were generally safe and well tolerated when administered using either an AI or PFS-NSD. It was comparable to AI vs. PFS-NSD in the pivotal cohort (9.5% vs. 13.2%).The majority of TEAEs were mild or moderate in severity and most resolved by the end of the study. One serious AE (seizure) led to study discontinuation in the PFS-NSD group of the study cohort, which was not considered related to treatment with etrolizumab.

Conclusions: The results of this PK equivalence study demonstrate that etrolizumab exposure is similar when SC is administered via either AI or PFS-NSD, and exposure parameters (AUC _last , C _max , AUC _inf ) was within the bioequivalence limits between instruments (80-125%). Furthermore, a single SC dose of etrolizumab administered via AI was generally well tolerated in healthy participants and was not associated with increased adverse events compared to PFS-NSD injection. This study also highlights the value of pilot cohorts in facilitating the design of pivotal PK equivalence studies, as data from pilot cohorts increased confidence in study design and minimized assumption bias.

Example 4
This example provides details regarding the PFS-NSD injection input.

There are multiple force definitions during the user's injection experience. The user actions are: 1) remove the needle cover, 2) push the plunger until the needle safety is activated. FIG. 22 shows different force definitions. Table 8 shows the forces and their limits. Table 9 shows that the injection force is within limits for both the 1 mL and 2.25 mL configurations.

There is a spring mechanism that pushes the liquid out for the autoinjector. Therefore, injection time is a relevant attribute. In certain embodiments, the injection time is from about 0.4 seconds to about 9 seconds. In certain embodiments, the injection time is about 5 seconds.

The autoinjector used in the formulations of the present disclosure provides favorable injection times and long-term stability.

Embodiments of the Disclosed Subject Matter From the foregoing description, it will be apparent that variations and modifications may be made to the presently disclosed subject matter for adaptation to various uses and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment 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 herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. incorporated into.

Claims (86)

A formulation comprising a monoclonal anti-integrin β7 antibody, wherein the anti-integrin β7 antibody has a concentration of at least about 100 mg/ml, the viscosity of the formulation is less than about 20 centipoise (cP) at 25° C., and the formulation is long-lasting. A formulation having stability. 2. The formulation of claim 1, wherein said anti-integrin β7 antibody is a humanized antibody. 2. The formulation of claim 1, wherein the formulation has a viscosity of about 7 cP at 25[deg.]C. 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. including
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) The formulation of any one of claims 1-3, wherein the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. 5. 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 variable region comprising the amino acid sequence shown in SEQ ID NO:9. The formulation described in section. 6. The anti-integrin β7 antibody according to any one of claims 1 to 5, comprising 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. formulation. 6. The anti-integrin β7 antibody according to any one of claims 1 to 5, comprising 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. formulation. The formulation of any one of claims 1-7, wherein said anti-integrin β7 antibody is etrolizumab. 9. The formulation of any one of claims 1-8, wherein the concentration of anti-integrin β7 antibody in the formulation is from about 100 mg/ml to about 220 mg/ml. 10. The formulation of any one of claims 1-9, wherein the concentration of anti-integrin β7 antibody in the formulation is 150 mg/mL or about 150 mg/ml. The formulation of any one of claims 1-10, wherein the formulation has a pH greater than 5.5. 13. The formulation of claim 12, wherein the formulation has a pH of 5.65-6.1. The formulation according to any one of claims 1 to 12, wherein the pH of said formulation is 5.8, 5.7-5.9 or 5.75-5.85. The formulation of any one of claims 1-13, further comprising a surfactant, wherein the concentration of said surfactant in said formulation is between 0.03% w/v and 0.06% w/v. . 15. The formulation of claim 14, wherein the concentration of said surfactant in said formulation is at or about 0.04% w/v. 16. A formulation according to claim 14 or 15, wherein said surfactant is polysorbate 20. 17. The formulation of any one of claims 1-16, further comprising arginine succinate. 18. The formulation of claim 17, wherein the concentration of arginine succinate in said formulation is from about 100 mM to about 300 mM. 19. The formulation of claim 17 or 18, wherein the concentration of arginine succinate in said formulation is from about 150 mM to about 300 mM. 20. The formulation of any one of claims 17-19, wherein the concentration of arginine succinate in said formulation is from about 150 mM to about 250 mM. The formulation of any one of claims 17-20, wherein the concentration of arginine succinate in said formulation is at or about 200 mM. The formulation of any one of claims 1-21, further comprising histidine. 23. The formulation of claim 22, wherein the concentration of histidine in said formulation is from about 5 mM to about 40 mM. 24. The formulation of claim 22 or 23, wherein the concentration of histidine in said formulation is at or about 20 mM. 25. The formulation of any one of claims 1-24, wherein said anti-integrin β7 antibody is stable at -20°C for at least about 7 years. 26. The formulation of any one of claims 1-25, wherein said anti-integrin β7 antibody is stable at 5°C for at least about 18 months. 27. The formulation of any one of claims 1-26, wherein said anti-integrin β7 antibody is stable at 5°C for at least about 2 years. 28. The formulation of any one of claims 1-27, wherein said anti-integrin β7 antibody is stable at room temperature for at least about one day. 29. The formulation of any one of claims 1-28, wherein said anti-integrin β7 antibody is stable at room temperature for up to about 1 month. A formulation comprising an anti-integrin β7 antibody, 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein the anti-integrin β7 antibody has a concentration of 150 mg/mL or about 150 mg/ml; An 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 ,
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) A formulation, wherein the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. 31. The formulation of claim 30, wherein said anti-integrin β7 antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. 32. The formulation of claim 30 or 31, wherein the anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11. 33. The formulation of claim 30 or 32, wherein the anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:12. 34. The formulation of any one of claims 30, 32 and 33, wherein said anti-integrin β7 antibody is etrolizumab. A formulation comprising an anti-integrin β7 antibody, 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0.04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein said anti-integrin β7 antibody comprises three light chain hypervariable regions (HVRs), HVR- comprising L1, HVR-L2 and HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) A formulation, wherein the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7. 36. The formulation of claim 35, wherein the anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11. 36. The formulation of claim 35, wherein said anti-integrin β7 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:10 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:12. The formulation of any one of claims 35-37, wherein said anti-integrin β7 antibody is etrolizumab. An article of manufacture comprising the formulation of any one of claims 1-38 and a subcutaneous administration device. 40. The article of manufacture of Claim 39, wherein said subcutaneous administration device is a needle safety device. 40. The article of manufacture of claim 39, wherein the needle safety device comprises a pre-filled syringe. 42. The article of manufacture of any one of claims 39-41, wherein said anti-integrin β7 antibody is stable in said subcutaneous administration device at 5°C for at least about 60 months, or at 25°C for at least about 3 months. . 43. The article of manufacture of claims 41 or 42, wherein the prefilled syringe comprises a glass barrel, plunger stopper, needle, and needle shield or tip cap. 44. The article of manufacture of claim 43, wherein the needle shield is a rigid needle shield. 45. The article of manufacture of claim 44, wherein the rigid needle shield comprises a low zinc content rubber compound. 46. The article of manufacture of claim 44 or 45, wherein the rigid needle shield comprises an elastomeric component and a rigid shield. 47. The article of manufacture of any one of claims 41-46, wherein the prefilled syringe is assembled into an autoinjector. 47. The article of manufacture of any one of claims 40-46, wherein the pre-filled syringe comprises silicone oil. 49. The article of manufacture of claim 48, wherein the amount of silicone oil in said pre-filled syringe is about 1 mg or less. 50. The article of manufacture of claim 48 or 49, wherein the amount of silicone oil in said pre-filled syringe is from about 0.1 mg to about 1 mg. 51. The article of manufacture of any one of claims 48-50, wherein the amount of silicone oil in the pre-filled syringe is from about 0.2 mg to about 0.6 mg. The article of manufacture of any one of claims 48-50, wherein the amount of silicone oil in said pre-filled syringe is about 0.5 mg to 0.9 mg. 53. The article of manufacture of any one of claims 40-52, wherein the needle safety device has an injection force of about 50 Newtons (N) or less. 54. The article of manufacture of any one of claims 40-53, wherein the needle safety device has an injection force of about 35 Newtons (N) or less. 55. The article of manufacture of any one of claims 39-54, comprising from about 0.5 mL to about 2.0 mL of said formulation. 56. The article of manufacture of any one of claims 39-55, comprising from about 0.5 mL to about 1.0 mL of said formulation. 57. The article of manufacture of any one of claims 39-56, comprising about 1.0 mL of said formulation. 57. The article of manufacture of any one of claims 39-56, comprising about 0.7 mL of said formulation. 56. The article of manufacture of any one of claims 39-55, comprising from about 1.0 mL to about 1.5 mL of said formulation. 60. The article of manufacture of any one of claims 39-55 and 59, comprising about 1.4 mL of said formulation. The article of manufacture of any one of claims 41-60, wherein the pre-filled syringe has a syringe capacity of 1 mL. The article of manufacture of any one of claims 41-60, wherein the pre-filled syringe has a syringe capacity of 2.25 mL. An article of manufacture comprising about 0.7 mL of formulation and a subcutaneous administration device,
(a) the formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0 04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein said anti-integrin β7 antibody comprises three light chain hypervariable regions (HVR), HVR-L1, HVR- comprising L2 and HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7;
(b) The article of manufacture, wherein said subcutaneous administration device is a needle safety device comprising a 1 mL pre-filled syringe having a 1 mL syringe capacity. 64. The article of manufacture of claim 63, wherein said anti-integrin β7 antibody is present in said formulation at a concentration of 150 mg/mL or about 150 mg/ml. An article of manufacture comprising about 1.4 mL of formulation and a subcutaneous administration device,
(a) the formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0 04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein said anti-integrin β7 antibody comprises three light chain hypervariable regions (HVR), HVR-L1, HVR- comprising L2 and HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7;
(b) The article of manufacture, wherein said subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe having a syringe capacity of 2.25 mL. 66. The article of manufacture of claim 65, wherein said anti-integrin β7 antibody is present in said formulation at a concentration of 150 mg/mL or about 150 mg/ml. An autoinjector comprising the article of manufacture of any one of claims 39-66. An autoinjector comprising an article of manufacture comprising about 0.7 mL of formulation and a subcutaneous administration device,
(a) the formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0 04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein said anti-integrin β7 antibody comprises three light chain hypervariable regions (HVR), HVR-L1, HVR- comprising L2 and HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7;
(b) The autoinjector, wherein said subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe having a 1 mL syringe capacity. 69. The autoinjector of claim 68, wherein said anti-integrin β7 antibody is present in said formulation at a concentration of 150 mg/mL or about 150 mg/ml. An autoinjector comprising an article of manufacture comprising about 1.4 mL of formulation and a subcutaneous administration device,
(a) the formulation contains 20 mM histidine buffer or about 20 mM histidine buffer (pH 5.8, or 5.7-5.9, or 5.75-5.85), 0.04% polysorbate 20 or about 0 04% polysorbate 20 and 200 mM arginine succinate or about 200 mM arginine succinate, wherein said anti-integrin β7 antibody comprises three light chain hypervariable regions (HVR), HVR-L1, HVR- comprising L2 and HVR-L3 and three heavy chain HVRs, HVR-H1, HVR-H2 and HVR-H3;
(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) said HVR-H2 comprises the amino acid sequence of SEQ ID NO:5;
(vi) the HVR-H3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:7;
(b) The autoinjector, wherein said subcutaneous administration device is a needle safety device with a 1 mL pre-filled syringe having a syringe capacity of 2.25 mL. 71. The autoinjector of claim 70, wherein said anti-integrin β7 antibody is present in said formulation at a concentration of 150 mg/mL or about 150 mg/ml. 39. A method of treating a gastrointestinal inflammatory disorder in a subject, comprising administering to said subject an effective amount of the formulation of any one of claims 1-38. 73. The method of claim 72, wherein said gastrointestinal inflammatory disorder is inflammatory bowel disease. 74. The method of claim 73, wherein said inflammatory bowel disease is ulcerative colitis or Crohn's disease. subcutaneously administering a formulation comprising an anti-integrin β7 antibody, comprising subcutaneously administering the article of manufacture of any one of claims 39-66 or the autoinjector of any one of claims 67-71 how to. 76. The method of claim 75, wherein said administering causes mild pain or no pain. 77. The method of claim 75 or 76, wherein said administering results in a transient and mild injection site reaction. 78. The method of any one of claims 75-77, wherein said total dose is administered, or at least 90% of said total dose is administered. 79. The method of any one of claims 75-78, wherein said administration provides equivalent exposure to etrolizumab compared to a pre-filled syringe with a needle safety device. 79. A method according to any one of claims 75-79, comprising a formulation according to any one of claims 1-37. A formulation according to any one of claims 1 to 38 for use in therapy. 67. The article of manufacture of any one of claims 39-66 for use in therapy. An autoinjector according to any one of claims 67-71 for use in therapy. A formulation according to any one of claims 1-38, an article of manufacture according to any one of claims 39-66, or claims 67-67 for use in treating a gastrointestinal inflammatory disorder in a subject. 72. The autoinjector of any one of Clauses 71. 85. The formulation, article of manufacture or auto-injection device of claim 84, wherein said gastrointestinal inflammatory disorder is inflammatory bowel disease. 86. A formulation, article of manufacture or autoinjector for use according to claim 85, wherein said inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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