BR122021011134B1 - STABLE LIQUID PHARMACEUTICAL FORMULATION COMPRISING AN ANTI-ALFA4BETA7 ANTIBODY AND MANUFACTURED ARTICLE - Google Patents

Abstract

Antibody formulations are described comprising a mixture of an anti-a4ß7 antibody, an antioxidant or chelator and at least one free amino acid. The disclosed formulations may have improved stability, reduced aggregate formation, or both. The present invention further provides a safe dosage regimen of these antibody formulations that is easy to follow and that results in a therapeutically effective amount of the anti-a4ß7 antibody in vivo.

Description

Split from BR112013028169-3, deposited on 05/02/2012. RELATED REQUESTS

[001] This application claims priority to provisional application US 61/544,054, filed October 6, 2011, and provisional application US 61/481,522, filed May 2, 2011. The complete contents of the previous applications are incorporated herein by reference .

SEQUENCE LISTING

[002] This application contains a Sequence Listing that was presented in ASCII format via EFS-Web and is incorporated by reference in its entirety. The said ASCII copy, created on April 30, 2012, is named 92596603.txt and is 16,986 bytes in size.

BASICS OF THE INVENTION

[003] Advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA techniques. Due to the fact that proteins are larger and more complex than traditional organic and inorganic drugs (that is, they have several functional groups in addition to complex three-dimensional structures), the formulation of said proteins presents special problems. For a protein to remain biologically active, a formulation must preserve the conformational integrity of at least one core amino acid sequence of the protein, while at the same time protecting the protein's various functional groups from degradation. Proteins can suffer from a lack of stability and monoclonal and polyclonal antibodies, in particular, can be relatively unstable (See, for example, Wang et al., J. Pharm Sci. 96:1-26 (2007)). A large number of formulation options are available and no single approach or system is available for all proteins. Several factors to consider have been reported (See, for example, Wang et al.).

[004] Numerous characteristics can affect the stability of a protein. In fact, even in the case of purified antibodies, antibody structures can be heterogeneous, which further complicates the formulation of said systems. Furthermore, excipients included in antibody formulations preferentially minimize any potential immune response.

[005] In the case of antibodies, preserving conformational integrity is even more important. Degradation pathways for proteins can involve chemical instability (i.e., any process that involves modification of the protein by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e., changes to the higher-order structure of the protein) . Chemical instability is manifested in, for example, deamidation, isomerization, hydrolysis, oxidation, fragmentation, beta glycan elimination or disulfide exchange. Chemical instability can result from denaturation, aggregation, precipitation or adsorption, for example. The four most common protein degradation pathways are protein fragmentation, aggregation, deamidation, and oxidation. Consequences of chemical or physical instability of therapeutic protein include a reduction in the effective dose administered, reduced safety of the therapy due to, for example, irritation or immunological reactivity, and, most frequently, manufacturing due to short half-life.

[006] Various publications have generally disclosed various methods for treating inflammatory bowel diseases and have provided dosage schedules for administering agents designed to treat inflammatory bowel disease. For example, WO 96/24673 discloses mucosal vascular addressins and treatment of diseases associated with leukocyte recruitment to the gastrointestinal tract as a result of leukocyte binding to MAdCAM-expressing cells. US 2005/0095238 describes methods for treating a disease associated with leukocyte infiltration of mucosal tissue and administering to a human an effective amount of a human or humanized immunoglobulin or antigen-binding fragment containing binding specificity for a4ß7 integrin. US 2005/0095238 further describes various doses (e.g., 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mg of immunoglobulin or fragment per kg of body weight) and various intervals between doses (7, 14, 21, 28, or 30 days). However, the aforementioned patents and publications do not disclose the specific formulations of the anti-a4ß7 antibody or the specific doses and dose regimens claimed herein. Importantly, the aforementioned patents do not disclose formulations, doses, and dose regimens that provide the treatment methods (supported by clinical study data) described and claimed here.

[007] The antibody formulations of the present invention may be useful for inhibiting leukocyte binding to MAdCAM-expressing cells and therefore assist in the treatment of inflammatory bowel diseases in patients. There is, therefore, an urgent need to discover appropriate dosages and dosing schedules of these compounds, and to develop formulations, preferably subcutaneous formulations, that generate stable, therapeutically effective blood levels of the antibody formulations over an extended period of time in a stable form. and convenient.

SUMMARY OF THE INVENTION

[008] The invention relates to the identification of an antioxidant or chelator and at least one amino acid as useful excipients to formulate anti-a4ß7 antibody formulations whose instability makes them susceptible to deamidation, oxidation, isomerization and/or aggregation. The formulation improves stability, reduces aggregate formation and slows degradation of the antibody therein.

[009] Thus, in a first aspect, the invention relates to a liquid pharmaceutical formulation comprising a mixture of an anti-a4ß7 antibody, an antioxidant or chelator and at least one free amino acid.

[0010] In some embodiments, the stable liquid pharmaceutical formulation has at least about 1.0% aggregate formation after 12 months at room temperature. The stable liquid pharmaceutical formulation may have less than about 0.2% aggregate formation after 12 months at room temperature.

[0011] In some embodiments, the antioxidant or chelator is citrate. In some embodiments the chelator is EDTA.

[0012] In some embodiments, the free amino acid of the formulation is histidine, alanine, arginine, glycine, glutamic acid, or any combination thereof. The formulation may comprise from about 50 mM to about 175 mM of the free amino acid. The formulation may comprise between about 100 mM and about 175 mM of the free amino acid. The free amino acid molar ratio to antibody molar ratio will be at least 250:1.

[0013] The formulation may also contain a surfactant. The surfactant may be polysorbate 20, polysorbate 80, a poloxamer, or any combination thereof.

[0014] In some embodiments, the molar ratio of antioxidant to surfactant is about 3:1 to about 156:1.

[0015] The formulation can have a pH between about 6.3 and about 7.0. The pH of the formulation can be between about 6.5 and about 6.8. The formulation may have a pH between about 6.1 and about 7.0, or between about 6.2 and 6.8.

[0016] In some embodiments, the stable liquid pharmaceutical formulation contains at least about 60 mg/ml to about 160 mg/ml anti-a4ß7 antibody. The formulation may contain at least about 160 mg/ml anti-a4ß7 antibody. The formulation may contain about 150 to about 180 mg/ml antibody or about 165 mg/ml antibody.

[0017] In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising at least about 60 mg/ml to about 160 mg/ml anti-a4ß7 antibody, a buffering agent and at least about 10 mM citrate . The buffering agent may be a histidine buffer.

[0018] In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising at least about 60 mg/ml to about 180 mg/ml anti-a4ß7 antibody, a buffering agent and at least about 5 mM citrate . The buffering agent may be a histidine buffer.

[0019] In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising at least about 160 mg/ml anti-a4ß7 antibody and at least about 10 mM citrate. The formulation may also contain polysorbate 80.

[0020] In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising about 160 mg/ml anti-a4ß7 antibody and at least about 5 mM citrate. The formulation may also contain polysorbate 80.

[0021] In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising a mixture of anti-a4ß7 antibody, citrate, histidine, arginine and polysorbate 80. The formulation may be present in a container, such as a vial, cartridge , syringe or autoinjector.

[0022] The anti-a4ß7 antibody in the stable liquid pharmaceutical formulation of the invention may be vedolizumab. The formulation of the invention can be for subcutaneous, intravenous or intramuscular administration.

[0023] In some aspects, the formulation can minimize immunogenicity of the anti-a4ß7 antibody.

[0024] In another aspect, the invention relates to a method for treating inflammatory bowel disease, comprising administering to a patient in need thereof the stable liquid pharmaceutical formulation described here. Administration may be subcutaneous administration. Administration can be self-administration.

[0025] In yet another aspect, the invention relates to an article of manufacture, comprising a container, a stable liquid pharmaceutical formulation described here, and instructions for its use.

[0026] In one aspect, the invention relates to a method for treating a human patient suffering from inflammatory bowel disease, wherein the method comprises the step of administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the following dosage regimen: (a) initial doses, e.g. , in an induction phase treatment regimen, of 165 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every other day for six doses; (b) followed in week six by a seventh and subsequent doses, for example, in a maintenance phase treatment regimen, of 165 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every two weeks or every four weeks as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein a humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[0027] In one aspect, the invention relates to a method for treating a human patient suffering from inflammatory bowel disease, wherein the method comprises the step of administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin, wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient in accordance with the following dosage regimen comprising an induction phase of intravenous doses and a maintenance phase of subcutaneous doses: (a) an initial intravenous dose of 300 mg of a humanized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion; (b) followed by a subsequent second intravenous dose of 300 mg of a humanized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion about two weeks after the initial dose; (c) followed beginning in week six by a third and subsequent doses of 165 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every week, every two weeks, every three weeks, or every four weeks as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[0028] In another aspect, the invention relates to a dosage regimen for the therapeutic treatment of inflammatory bowel disease, wherein the dosage regimen comprises the step of: administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin , wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to a subcutaneous or intramuscular dosage regimen that maintains a mean steady-state serum concentration of the immunoglobulin or antigen-binding fragment thereof of about from 9 to about 13 µg/mL; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[0029] In another aspect, the invention relates to a dosage regimen for the therapeutic treatment of inflammatory bowel disease, wherein the dosage regimen comprises the step of: administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin , wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to a subcutaneous or intramuscular dosage regimen that maintains a mean steady-state serum concentration of a humanized immunoglobulin or antigen-binding fragment thereof from about 35 to about 40 µg/mL; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[0030] In another aspect, the invention relates to a method for treating a human patient suffering from inflammatory bowel disease, wherein the method comprises the step of: administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin , wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient in accordance with the following dosage regimen: (a) a plurality of induction phase doses of a humanized immunoglobulin or antigen-binding fragment thereof sufficient to achieve a significant mean serum concentration of about 20 to about 30 µg/mL of a humanized immunoglobulin or antigen-binding fragment thereof by about six weeks of initial dosing; (b) followed by a plurality of maintenance phase doses of a humanized immunoglobulin or antigen-binding fragment thereof as necessary to maintain a mean steady-state serum concentration of about 9 to about 13 µg/mL or about 35 to 40 µg/mL of immunoglobulin or antigen-binding fragment thereof; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO:13; heavy chain: CDR1 SEQ ID NO:8, CDR2 SEQ ID NO:9, CDR3 SEQ ID NO:10.

[0031] In some aspects, the formulation, treatment method, dose and/or dose regimen ensures minimal probability that a patient will develop antibodies reactive to the anti-a4ß7 antibody.

[0032] The patient may have had a lack of adequate response to, loss of response to or was intolerant to treatment with at least one of an immunomodulator, a tumor necrosis factor alpha (TNF-a) antagonist or combinations thereof.

[0033] The inflammatory bowel disease can be Crohn's disease or ulcerative colitis. Inflammatory bowel disease can be moderate to severely active ulcerative colitis.

[0034] The dosage regimen may result in mucosal healing in patients suffering from moderately to severely active ulcerative colitis.

[0035] The patient may have previously received treatment with at least one corticosteroid for inflammatory bowel disease. The patient may concomitantly receive treatment with at least one corticosteroid for inflammatory bowel disease. The dosage regimen may result in a reduction, elimination, or reduction and elimination of the patient's corticosteroid use.

[0036] In some aspects, the humanized immunoglobulin or antigen-binding fragment thereof is administered in a final dosage form at a concentration of between about 1.0 mg/ml to about 1.4 mg/ml. The humanized immunoglobulin or antigen-binding fragment thereof can be administered in a final dosage form of about 1.2 mg/ml.

[0037] In some aspects, the humanized immunoglobulin or antigen-binding fragment is administered in a final dosage form containing an amount of anti-a4ß7 antibody between about 70 to about 250 mg, between about 90 to about 200 mg, between about 150 to about 180 mg, or at least 160 mg.

[0038] In some aspects, the dosage regimen does not alter the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving said treatment.

[0039] The patient may be a person 65 years of age or older and does not require any adjustment of the dose regimen.

[0040] In some aspects the method of treatment with anti-a4ß7 antibody formulation, the dose, or dose regimen can minimize immunogenicity of the anti-a4ß7 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is an illustration of a nucleotide sequence (SEQ ID NO:1) encoding the heavy chain of a humanized anti-a4ß7 immunoglobulin, and the deduced amino acid sequence of the heavy chain (SEQ ID NO:2). The nucleotide sequence contains cloning sites (lower case), Kozak sequence (upper case, nucleotides 18-23 of SEQ ID NO:1) and leader sequence (lower case, nucleotides 24-86 of SEQ ID NO:1) in the 5' end of the heavy chain. The open reading region of the nucleotide sequence is nucleotides 24-1433 of SEQ ID NO:1.

[0042] FIG. 2 is an illustration of a nucleotide sequence (SEQ ID NO: 3) encoding the light chain of a humanized immunoglobulin referred to herein as vedolizumab, and the deduced amino acid sequence (SEQ ID NO: 4) of the light chain. The nucleotide sequence contains cloning sites (lower case), Kozak sequence (upper case, nucleotides 18-23 of SEQ ID NO:3) and leader sequence (lower case, nucleotides 24-80 of SEQ ID NO:3) at the end 5' of the heavy chain. The open reading region of the nucleotide sequence is nucleotides 24-737 of SEQ ID NO:3.

[0043] FIG. 3 is an alignment of the amino acid sequences of (A) the mature humanized light chain (amino acids 20-238 of SEQ ID NO:4) of a humanized immunoglobulin referenced herein as vedolizumab and (B) the mature humanized light chain of a humanized immunoglobulin referenced here as LDP-02 (SEQ ID NO:5). (Regarding LDP-02, see, WO 98/06248 and Feagan et al., N. Eng. J. Med. 352:2499-2507 (2005)). Feagan et al. describes a clinical study of LDP-02, but in the article they refer to LDP-02 as MLN02.) The alignment illustrates that the amino acid sequences of the light chains of vedolizumab and LDP-02 differ at positions 114 and 115 of the mature light chains .

[0044] FIG. 4 is an alignment of amino acid sequences of (A) a generic human kappa light chain constant region (SEQ ID NO:6) and (B) a generic murine kappa light chain constant region (SEQ ID NO:7). The amino acid residues Thr and Val (which are present at positions 114 and 115 of the mature vedolizumab light chain (amino acids 133 and 134 of SEQ ID NO:4)) are present in the constant region of the human kappa light chain, whereas the amino acid residues of amino acid Ala and Asp (which are present at positions 114 and 115 of the mature LDP-02 light chain (SEQ ID NO:5)) are present in the constant region of the mouse kappa light chain.

[0045] FIG. 5 is a vector map pLKTOK38D (further referred to as pTOK38MLN02-TV), which encodes the humanized heavy chain and the humanized light chain of MLN02, and is suitable for production in CHO cells. (See, US patent application publication 2004/0033561 A1 disclosing pLKTOK38. pLKTOK38D is a variant of pLKTOK38 in which the restriction sites in the map flank the sequence encoding the light chain variable region.)

[0046] FIG. 6 shows the slope of SEC aggregate formation (% per day) as a result of changes to protein concentration, pH and surfactant:protein molar ratio. In a pH range of 6.0 to 6.5, aggregate formation was similar for the formulation with a polysorbate 80:protein molar ratio of 0.7 to 1.5.

[0047] FIG. 7 is a graph showing that at polysorbate 80:protein molar ratios greater than 1.5, the rate of aggregate formation increases as pH increases.

[0048] FIG. 8 is a graph showing the effect of excipients on aggregate formation. 25mM citrate, 5mM citrate, 5mM EDTA, 25mM cysteine or 5mM cysteine were added to the formulations. All three excipients reduced aggregate formation.

[0049] FIG. 9 is a set of graphs showing the reduction in aggregate formation with the presence of 25 mM citrate in the formulation and a correlation between increased protein concentration and increased rate of aggregate formation.

[0050] FIG. 10 is a graph showing the results of CEX species degradation at 40°C. The data show the influence of pH change on CEX degradation.

[0051] FIG. 11 is a graph that shows the effect of temperature on the pH of the formulations. The pH of formulations containing histidine decreases with temperature, while the pH of citrate formulations is not affected by temperature.

[0052] FIG. 12 is a graph showing the percentage of major CEX isoform over a twelve month period. Formulations having a pH of 6.0-6.2 showed about 1-2% less major isoform than formulations having a pH of 6.3-6.4.

[0053] FIG. 13 shows a set of graphs that demonstrate that viscosity is mainly affected by protein concentration and pH. Additions of sucrose, histidine and arginine have been shown to have a minor effect on formulation viscosity.

[0054] FIG. 14 shows the amino acid sequences of (A) human mature GM607'CL antibody kappa light chain variable region and (B) human 21/28'CL heavy chain variable region.

[0055] FIG. 15 shows components of a protein product in a prefilled syringe.

[0056] FIGS. 16A-B show the effect of (A) protein concentration and (B) viscosity on the injection force of various syringes tested.

[0057] FIG. 17 (A) shows the initial sliding force as a function of protein concentration and needle size. FIG. 17(B) shows the initial sliding force for each syringe manufacturer and needle size.

[0058] FIG. 18 shows the absorption profile of vedolizumab. The graph shows that intramuscular and subcutaneous dose concentrations generally overlap. There are no apparent gross differences in the absorption profiles of these routes of administration.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The invention relates to a pharmaceutical formulation comprising anti-a4ß7 antibodies. The pharmaceutical formulation may be a mixture comprising an antioxidant or chelator (e.g., citrate), anti-a4ß7 antibody and a free amino acid. The pharmaceutical formulation can be in a solid or liquid form. Definitions

[0060] The term “pharmaceutical formulation” refers to a preparation that contains an anti-a4ß7 antibody in such a way as to allow biological activity to be effective, and that does not contain additional components that are unacceptably toxic to a subject to whom the formulation could be administered.

[0061] A “stable” formulation is one in which the antibody therein substantially retains its physical stability and/or its chemical stability and/or its biological activity in storage. In one aspect, the formulation substantially retains its physical and chemical stability, as well as its biological activity in storage. The storage period is generally selected based on the shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, the liquid formulation is stable at about 40°C for at least about 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. In another aspect, the lyophilized formulation is stable at about 40°C for at least about 2-4 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, or at least about 18 months. The liquid and/or lyophilized formulation in another aspect is stable at about 5°C and/or 25°C for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, or at least about 36 months; and/or were at about -20°C and/or -70°C for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. Furthermore, the liquid formulation may, in some embodiments, be stable after freezing (at, for example, -80°C) and thawing, for example, after 1, 2 or 3 cycles of freezing and thawing.

[0062] The stability of a liquid formulation can be assessed qualitatively and/or quantitatively in a variety of different ways, including assessment of dimer, multimer, and/or aggregate formation (e.g., using size exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), analytical ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), static light scattering, scattering multi-angle laser light (MALLS), flow-based microscopic imaging, electronic impedance counting (coulter), light obscuration in another liquid particle counting system, by turbidity measurement, and/or by visual inspection); by assessment of charge heterogeneity using cation exchange chromatography (CEX), isoelectric focusing (IEF), e.g. capillary technique (cIEF), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE or SEC analysis to compare fragmented, intact, and multimeric (i.e., dimeric, trimeric, etc.) antibody; peptide map analysis (e.g., tryptic or LYS-C); assessment of biological activity or antigen-binding function; and the like. The stability of a solid-state formulation can further be assessed qualitatively and/or quantitatively in a variety of different ways, including direct tests such as crystal structure identification by X-ray powder diffraction (XRPD); evaluating the structure of the antibody in the solid state using Fourier Transform Infrared Spectroscopy (FTIR); and measuring thermal transitions in the freeze-dried solid (melting, glass transition, etc.) using differential scanning calorimetry (DSC) and indirect tests such as measuring moisture content by Karl Fisher test, for example, to extrapolate the probability of chemical instability through hydrolysis. Instability may involve any one or more of: aggregation (e.g., soluble non-covalent aggregation, soluble covalent aggregation (e.g., disulfide bond rearrangement/shuffling), insoluble aggregation), deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), nicking/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, N-terminal extension, C-terminal processing, glycosylation differences , and the like.

[0063] A “deamidated” monoclonal antibody is one in which one or more asparagine or glutamine residues thereof have been derived, for example, to an aspartic acid or an isoaspartic acid.

[0064] An antibody that is “susceptible to deamidation” is one comprising one or more residues that have been shown to be prone to deamidation.

[0065] An antibody that is “susceptible to oxidation” is an antibody comprising one or more residues that have been shown to be prone to oxidation.

[0066] An antibody that is “susceptible to aggregation” is one that has been shown to aggregate with other antibody molecules, especially upon freezing, heating, drying, reconstitution and/or shaking.

[0067] An antibody that is “susceptible to fragmentation” is one that has been shown to be cleaved into two or more fragments, for example, in a hinge region thereof.

[0068] By “reduce deamidation, oxidation, aggregation, or fragmentation” is intended to mean preventing or reducing (e.g., by 80%, 60%, 50%, 40%, 30%, 20%, or 10% of) the amount of deamidation, aggregation, or fragmentation with respect to the monoclonal antibody formulated at a different pH or in a different buffer.

[0069] An “aggregate”, “SEC aggregate”, or “soluble aggregate” is more than one and less than or equal to ten antibody proteins and/or fragments associated together by covalent, ionic or hydrophobic interactions to form a larger protein body.

[0070] An “insoluble aggregate” or “particle” is larger than ten antibody proteins and/or fragments associated by covalent, ionic or hydrophobic interactions to form a larger protein body.

[0071] As used herein, “biological activity” of a monoclonal antibody refers to the ability of the antibody to bind to the antigen and result in a measurable biological response that can be measured in vitro or in vivo. Said activity can be antagonist or agonist.

[0072] The cell surface molecule, “a4ß7 integrin,” or “a4ß7,” is a heterodimer of an a4 chain (CD49D, ITGA4) and a ß chain? (ITGB7). Each chain can form a heterodimer with an alternative integrin chain, to form, for example, a4ßi or aEß?. Human genes a4 and ß? (GenBank (National Center for Biotechnology Information, Bethesda, MD) RefSeq accession numbers NM_000885 and NM_000889, respectively) are expressed by B and T lymphocytes, particularly CD4+ memory lymphocytes. Typical of many integrins, a4ß7 can exist in a resting or activated state. Ligands for a4ß7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., MAdCAM-1)).

[0073] As used here, a human immunoglobulin or antigen-binding fragment thereof that has “binding specificity for the a4ß7 complex” binds to a4ß7, but not to a4ß1 or aEB7.

[0074] As used here, an “isotonic” formulation has substantially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing-type osmometer, for example.

[0075] As used herein, “buffering agent” refers to a buffer that resists changes in pH by the action of its conjugated acid-base components. The buffering agent may be present in a liquid or solid formulation of the invention. In some embodiments, the buffering agent of this invention adjusts the pH of the formulation to about 5.0 to about 7.5, to about pH 5.5 to about 7.5, at about pH 6.0 to about 7.0, or at a pH of about 6.3 to about 6.5. In one aspect, examples of buffering agents that alone or in combination will control pH in the range of 5.0 to 7.5 include acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, cacodylate, 2-[ N-morpholino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris [hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and other organic acid buffers. In another aspect, the buffering agent here is histidine or citrate.

[0076] A “histidine buffer” is a buffer comprising histidine ions. Examples of histidine buffers include solutions of histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The histidine buffer or histidine-HCl buffer has a pH between about pH 5.5 to about pH 7.0, between about pH 6.1 to about 6.9, or about pH 6.5.

[0077] A “citrate buffer” is a buffer comprising citrate ions. Examples of citrate buffers include solutions of sodium citrate, ammonium citrate, calcium citrate, and potassium citrate. Citrate buffer has a pH of about 3.0 to about 6.2, about pH 5.5 to 6.5, about pH 6.1 to about 6.5, about pH 6.1, about of pH 6.2, or about pH 6.5.

[0078] A "saccharide" here is a compound having a general formula (CH2O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of saccharides here include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol , lactitol, isomaltulose, and the like. A saccharide can be a lyoprotectant. In another aspect, the saccharide here is a non-reducing disaccharide, such as sucrose.

[0079] Here, a “surfactant” refers to an agent that reduces the surface tension of a liquid. In one aspect, the surfactant is a nonionic surfactant. Examples of surfactants herein include polysorbate (polyoxyethylene sorbitan monolaurate, e.g., polysorbate 20 and polysorbate 80); TRITON (t-Octylphenoxypolyethoxyethanol, nonionic detergent, Union Carbide subsidiary of Dow Chemical Co., Midland MI); sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; sodium octyl glycoside; laurel-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl, or disodium methyl oleyl taurate; sorbitan monopalmitate; and the MONAQUAT series (Mona Industries, Inc., Paterson, N.J.); polyethyl glycol (PEG), polypropylene glycol (PPG), and copolymers of polyxyethylene and polyxypropylene glycol (e.g., Pluronics/Polixamer, PF68, etc.); etc. In another aspect, the surfactant here is polysorbate 80.

[0080] The term “chelator” refers to someone who binds to an atom through more than one bond. In one aspect, examples of chelators herein include citrate, ethylenediaminetetraacetic acid, ethyleneglycoltetraacetic acid (EGTA), dimercaprol, diethylenetriaminepentaacetic acid, and N,N-bis(carboxymethyl)glycine. In another aspect, the chelator is citrate or EDTA.

[0081] The term “antioxidant” refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants here include citrate, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, phosphonate compounds, for example, etidronic acid, deferoxamine and malate.

[0082] The term “antibody” here is used in the broadest sense and specifically covers complete monoclonal antibodies, immunoglobulins, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two full-length antibodies, e.g. each on a different antigen or epitope, and individual antigen-binding fragments, including dAbs, scFv, Fab, F(ab)'2, Fab', including human and humanized antibodies from non-human species and recombinant antigen-binding forms such as monobodies and diabodies.

[0083] Amounts and molar ratios of anti-a4ß7 antibody to other excipients described here are calculated assuming an approximate molecule weight of about 150,000 daltons for the antibody. The actual molecular weight of the antibody may differ from 150,000 daltons depending on the amino acid composition or post-translational modification, for example, as dependent on the cell line used to express the antibody. The actual molecular weight of the antibody may be +/- 5% of 150,000 daltons.

[0084] The term “human antibody” includes an antibody that has a sequence that is derived from a human germline immunoglobulin sequence, such as an antibody derived from transgenic mice containing human immunoglobulin genes (e.g., genetically modified XENOMOUSE mice (Abgenix, Fremont, CA), HUMAB-MOUSE®, KIRIN TC MOUSE™ transchromosomal mice, KMMOUSE® (MEDAREX, Princeton, NJ)), human phage display libraries, human Myeloma cells, or human B cells.

[0085] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants that may arise during monoclonal antibody production, said variants generally being present in smaller quantities. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the characteristics of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by recombinant DNA methods (see , e.g., US patent 4,816,567). "Monoclonal antibodies" can further be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 -597 (1991), for example.

[0086] Monoclonal antibodies herein specifically include “chimeric” antibodies in which a part of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass. or, while the remainder of the chains are identical with or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of said antibodies, provided that they exhibit the desired biological activity (US patent 4,816 567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest here include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0087] “Antigen-binding fragments” of a humanized immunoglobulin prepared in the formulation of the invention comprise at least the variable regions of heavy and/or light chains of an anti-a4ß7 antibody. For example, a vedolizumab antigen-binding fragment comprises amino acid residues 20-131 of the humanized light chain sequence of SEQ ID NO:4. Examples of said antigen-binding fragments include Fab fragments, Fab' fragments, scFv and F(ab')2 fragments of a humanized immunoglobulin known in the art. Antigen-binding fragments of a humanized immunoglobulin of the invention can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can be used to generate Fab or F(ab')2 fragments, respectively. Antibodies can further be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a recombinant construct encoding the heavy chain of an F(ab')2 fragment can be designed to include DNA sequences encoding the CHI domain and hinge region of the heavy chain. In one aspect, antigen-binding fragments inhibit the binding of an a4ß7 integrin to one or more of its ligands (e.g., the mucosal MAdCAM addressin (e.g., MAdCAM-1), fibronectin).

[0088] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a unique antigen-binding site, and a residual “Fc” fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment generates an F(ab')2 fragment that has two binding sites and is still capable of cross-linking with an antigen.

[0089] “Fv” is an antibody fragment consisting of a dimer of a heavy chain variable domain and a light chain variable domain in non-covalent association.

[0090] The Fab fragment still contains the light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of some residues at the carboxy terminus of the CH1 heavy chain domain including one or more cysteines of the antibody hinge region. Fab'-SH is the designation here for Fab' in which the cysteine residues of the constant domains have at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are still known.

[0091] “Single-chain Fv” or “scFv” antibody fragments comprise the antibody VH and VL domains, where these domains are present in a single polypeptide chain. In one aspect, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0092] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, whose fragments comprise a variable chain domain (VH) connected to a variable light domain (VL) on the same polypeptide chain (VH -VL). Using a linker that is too small to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. academic Sci. USA, 90:6444-6448 (1993).

[0093] A “complete antibody” is one that comprises an antigen-binding variable region as well as a constant light chain (CL) domain and constant heavy chain domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In one aspect, the complete antibody has one or more effector functions.

[0094] An “amino acid sequence variant” antibody here is an antibody with an amino acid sequence that differs from the antibody of the parent species. Ordinarily, amino acid sequence variants will possess at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the antibody of the main species. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions within or adjacent to the amino acid sequence of the parent species antibody, but retain antigen-binding activity. Variations in the sequence of the constant regions of the antibody will have less effect on antigen-binding activity than variations in the variable regions. In the variable regions, amino acid sequence variants will be at least about 90% homologous, at least about 95% homologous, at least about 97% homologous, at least about 98% homologous, or at least about 99% homologous. with the antibody of the parent species.

[0095] “Homology” is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to obtain maximum percentage homology. Methods and computer programs for alignment are well known in the art.

[0096] A “therapeutic monoclonal antibody” is an antibody used for therapy of a human subject. Therapeutic monoclonal antibodies disclosed herein include anti-a4ß7 antibodies.

[0097] A “variant glycosylation” antibody here is an antibody with one or more carbohydrate moieties linked to it that differ from one or more carbohydrate moieties linked to an antibody of the main species. Examples of glycosylation variants herein include antibody with a G1 or G2 oligosaccharide structure, rather than a G0 oligosaccharide structure, linked to an Fc region thereof, antibody with one or two carbohydrate moieties linked to one or two chains thereof. , non-carbohydrate antibody linked to one or two heavy chains of the antibody, etc., and combinations of glycosylation changes.

[0098] Antibody “effector functions” refer to those antibody activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

[0099] Depending on the amino acid sequence of the constant domain of their heavy chains, complete antibodies can be designated as being of different “classes”. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into “subclasses” (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, d, e, Y, and µ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[00100] The “light chains” of antibodies from any vertebrate species can be designated as one of two clearly distinct types, called kappa (K) and lambda (À), based on the amino acid sequences of their constant domains.

[00101] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils , and macrophages) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. The primary cells to mediate ADCC, NK cells, express only FcYRIII, whereas monocytes express FCYRI, FCYRII, and FCYRIII. Expression of FcR in hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in US patents 5,500,362 or 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652656 (1998).

[00102] The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. In one aspect, the FcR is a human native sequence FcR. In another aspect, the FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FCYRI, FCYRII, and FCYRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. Activation receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Receptor inhibition FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, review in M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:33-41 (1995). Other FcRs, including those identified in the future, are included by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 ( 1994)).

[00103] The term “hypervariable region” when used here refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region generally comprises amino acid residues of a “complementarity determining region” or “CDR” (e.g., residues 24-34 (L1), 5056 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, Md. (1991)) and/or those residues of a “hypervariable loop” (e.g., residues 2632 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework region” or “FR” residues are those whose variable domain residues in addition to hypervariable region residues are defined herein. The hypervariable region or CDRs thereof can be transferred from one antibody chain to another or to another protein to impart antigen-binding specificity to the resulting antibody (composite) or binding protein.

[00104] “Humanized” forms of non-human antibodies (e.g., rodents) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate containing the desired specificity, affinity and capacity. In some cases, residues from the framework region (FR) of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient's antibody or in the donor's antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a sequence of human immunoglobulin. The humanized antibody will optionally further comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[00105] A “mature affinity” antibody is one with one or more changes in one or more hypervariable regions thereof that result in an improvement in the affinity of the antibody to the antigen, compared to a relative antibody that does not have those changes. In one aspect, mature affinity antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity mature antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or structural residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[00106] An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight protein as determined by the Lowry method, and alternatively, to greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence by use of a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver dye. Isolated antibody includes the antibody in situ within the recombinant cell since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[00107] “Treatment” refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who are already ill as well as those in whom the illness or its recurrence must be prevented. Thus, the patient being treated here may have been diagnosed as having the disease or may be predisposed or susceptible to the disease. The terms “patient” and “subject” are used interchangeably here.

[00108] The antibody that is formulated is substantially pure and desirably substantially homogeneous (i.e., free from contaminating proteins, etc.). "Substantially pure" antibody means a composition comprising at least about 90% antibody by weight, based on total protein weight, alternatively, at least about 95% or 97% by weight. “Substantially homogeneous” antibody means a composition comprising protein in which at least about 99% by weight of protein is specific antibody, e.g., anti-a4ß7 antibody, based on total protein weight.

[00109] “Clinical remission” as used herein with reference to subjects with ulcerative colitis refers to a complete Mayo score of 2 or fewer points and no individual subscore greater than 1 point. “Clinical remission” of Crohn's disease refers to a CDAI score of 150 points or less.

[00110] A “clinical response” as used herein with reference to subjects with ulcerative colitis refers to a reduction in full Mayo score of 3 or more points and 30% of baseline, (or a partial Mayo score of 2 or more points) more points and 25% or more of baseline if the full Mayo score was not performed at the visit) with an accompanying reduction in rectal bleeding subscore of 1 or more points or absolute rectal bleeding score of 1 or fewer points. A “clinical response” as used here with respect to subjects with Crohn's disease refers to a reduction of 70 points or more in the CDAI score from baseline (week 0).

[00111] “Mucosal healing” as used herein with respect to subjects with ulcerative colitis refers to an endoscopic subscore of 1 point or less.

[00112] As used herein, “treatment failure” refers to a worsening of the disease, a need for rescue medications or surgical intervention to treat ulcerative colitis or Crohn's disease. A rescue medication is any new medication or any increase in the dose of a baseline medication required to treat new or unresolved symptoms of ulcerative colitis or Crohn's disease (other than antidiarrheals or chronic diarrhea).

Formulations

[00113] As described here, it has been discovered that anti-a4ß7 antibodies are more stable when formulated with an antioxidant or chelator. Furthermore, as described herein, anti-a4ß7 antibodies can be formulated to reduce aggregate formation (e.g., the amount of polysorbate 80 in the formulation can be reduced). For example, formulations comprising citrate or EDTA and anti-a4ß7 antibodies reduce the rate of antibody aggregate formation during storage. Formulations can also be stored without oxygen to reduce aggregate formation. In one embodiment, the formulation of an antibody aggregate formation of less than about 2.5% at 25°C after 12 months. In one embodiment, the formulation of an antibody aggregate formation of less than about 2.0% at 25°C after 12 months. In one embodiment, the formulation of an antibody aggregate formation of less than about 1.6% at 25°C after 12 months. In one embodiment, the formulation of an antibody aggregate formation of less than about 1.3% at 25°C after 12 months. In one embodiment, the formulation of an antibody aggregate formation of less than about 1.0% at 25°C after 12 months. In another embodiment, the formulation has an antibody aggregate formation of less than about 0.5% at 5°C after 12 months. In another embodiment, the formulation has an antibody aggregate formation of less than about 0.3% at 5°C after 12 months.

[00114] The present invention provides, in a first aspect, a stable anti-a4ß7 antibody formulation. The formulation comprises an anti-a4ß7 antibody and an antioxidant or chelator. The formulation further comprises a buffering agent which may be one or more free amino acids. The formulation may optionally further comprise a surfactant. The antibody in the formulation may be a complete antibody or an antigen-binding fragment thereof, such as a Fab, Fv, scFv, Fab' or F(ab')2 fragment.

[00115] Aggregate formation can be reduced by removing oxygen from the formulation. Alternatively, the formulation may contain an antioxidant or chelator. In one aspect, exemplary antioxidants and chelators that may be included in the formulation include lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycoltetraacetic acid (EGTA), dimercaprol, diethylenetriaminepentaacetic acid, and N. ,N-bis(carboxymethyl)glycine, phosphonate compounds, e.g. etidronic acid, desferoxamine, malate and citrate. Some antioxidants and chelators can reduce the rate of aggregate formation during formulation storage. In another aspect, the chelator and/or antioxidant is citrate or EDTA. Exemplary chelator concentrations for liquid formulations are in the range of about more than 0 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 15 mM, about 10 mM to about of 25 mM, and about 20 to about 30 mM. In another aspect, the concentration of the chelator is from about 0 mM to about 30 mM. In one embodiment, the chelator and/or antioxidant is citrate, and the concentration of citrate is from about 0 mM to about 15 mM, about 0 mM to about 10 mM, or about 0 mM to about 5 mM. .

[00116] The formulation may contain any desired or free amino acid, which may be in L-form, D-form or any desired mixture of these forms. In one aspect, free amino acids that may be included in the formulation include, for example, histidine, alanine, arginine, glycine, glutamic acid, serine, lysine, tryptophan, valine, cysteine and combinations thereof. Some amino acids can stabilize proteins against degradation during manufacturing, drying, freeze-drying and/or storage, for example, through hydrogen bonds, salt bridges, antioxidant properties, or hydrophobic interactions or by exclusion from the protein surface. Amino acids can act as tonicity modifiers or can act to reduce the viscosity of the formulation. In another aspect, free amino acids, such as histidine and arginine, can act as lyoprotectants, and do not crystallize when lyophilized as components of the formulation. Free amino acids, such as glutamic acid and histidine, alone or in combination, can act as buffering agents in aqueous solution in the pH range of 5 to 7.5. In yet another aspect, the formulation contains histidine, arginine, or a combination of histidine and arginine. In yet another aspect, free amino acid concentrations for liquid formulations are in the range of about 9 mM to about 0.5 M, e.g., from about 10 mM to about 90 mM, about 10 mM to about 75 mM. mM, about 10 mM to about 40 mM, about 25 mM to about 50 mM, about 15 mM to about 300 mM, about 20 mM to about 200 mM, about 25 mM to about 150 mM, about 50 mM to about 75 mM, about 50 mM to about 120 mM, about 50 to about 150 mM, or about 50 mM or about 125 mM.

[00117] The formulation may optionally also contain at least one surfactant, for example, to control soluble or insoluble aggregate formation. In one aspect, the surfactant is a nonionic surfactant. In another aspect, the surfactant is an ionic surfactant. Exemplary surfactants that may be included in the formulation include, for example, polysorbate 20, polysorbate 80, a poloxamer (Pluronic®) and combinations thereof. When present, the surfactant is generally included in an amount that reduces the formation of insoluble antibody aggregates, e.g., during bottling, freezing, drying, lyophilization, and/or reconstitution, in the presence of silicone, filled vials, prefilled syringes, and/or or cartridges. The surfactant concentration is generally from about 0.0001% to about 1.0%, from about 0.01% to about 0.5%, for example, about 0.05%, 0.1% , 0.15%, 0.20%, 0.3%, 0.4%, or 0.5% (w/v). Higher concentrations of surfactant, e.g., polysorbate 80, may lead to the formation of more SEC aggregate. Reducing polysorbate 80 concentration can reduce SEC aggregate formation in storage. In one aspect, the surfactant:antibody molar ratio is from about 0.7:1 to about 2.0:1. In another aspect, the surfactant:antibody molar ratio is 1.5:1.

[00118] One embodiment of an anti-a4ß7 antibody formulation contains a high concentration of the anti-a4ß7 antibody. For example, in one embodiment, the liquid formulations may comprise at least about 60 mg/ml, at least about 70 mg/ml, at least about 80 mg/ml, at least about 90 mg/ml, at least about about 100 mg/ml, at least about 110 mg/ml, at least about 120 mg/ml, at least about 130 mg/ml, at least about 140 mg/ml, at least about 150 mg/ml ml, at least about 170 mg/ml, at least about 190 mg/ml, at least about 160 mg/ml, at least about 180 mg/ml, at least about 200 mg/ml, at least about from 250 mg/ml, mg/ml from about 60 mg/ml to about 190 mg/ml, from about 60 mg/ml to about 170 mg/ml anti-a4ß7 antibody, from about 150 mg/ml ml to about 180 mg/ml, or about 160 mg/ml or about 165 mg/ml anti-a4ß7 antibody. Alternatively, in another aspect, the liquid formulations may comprise at least about 154 mg/ml, at least about 176 mg/ml.

[00119] The formulation can be a liquid or a solid. Liquid formulations are aqueous solutions or suspensions, prepared in an appropriate aqueous solvent, such as water or an aqueous/organic mixture, such as mixtures of alcohol and water. Liquid formulations have a pH between about 5.5 and about 7.5, between about 6.0 and 7.3, between about 6.0 and about 7.0, between about 6.0 and 6.5, between about 6.0 and 6.3, between about 6.3 and 7.1, or between about 6.4 and 7.0, or between 6.3 and 6.8, like about of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9. Liquid formulations can be kept at room temperature, refrigerated (e.g., 2-8oC), or frozen (e.g., -20oC or -70oC) for storage.

[00120] A solid formulation can be prepared in any appropriate way and can be in the form of a cake or powder, for example, with the addition of a lyoprotectant. In one aspect, the solid formulation is prepared by drying a liquid formulation as described herein, for example, by lyophilization or spray drying. When the formulation is a solid formulation, the formulation may have a moisture content of no more than about 5%, no more than about 4.5%, no more than about 4%, no more than about about 3.5%, no more than about 3%, no more than about 2.5%, no more than about 2%, no more than about 1.5%, no more than than about 1%, or is substantially anhydrous. A solid formulation can be dissolved, i.e. reconstituted, in a suitable medium or solvent to become a liquid suitable for administration. Suitable solvents for reconstitution of the solid formulation include water, isotonic saline, buffer, e.g., phosphate buffer saline, Ringer's solution (lactate or dextrose), minimal essential medium, alcohol/aqueous solutions, dextrose solution, etc. The amount of solvent may result in a therapeutic protein concentration greater than, equal to, or less than the concentration before drying. In another aspect, the reconstituted anti-a4ß7 antibody concentration is the same concentration as in the pre-drying liquid formulation.

[00121] The formulation may be sterile, and this may be achieved in accordance with procedures known to those skilled in the art for generating pharmaceutical formulations suitable for administration to human subjects, before or after preparation of the formulation. The formulation may be sterilized as a liquid, for example, before drying and/or after reconstitution by small-pore filtration, aseptic processing, or exposure to ultraviolet radiation. Filter pore sizes can be 0.1 µm or 0.2 µm to filter microorganisms or 10 to 20 nm to filter viral particles. Alternatively, or additionally, the dry formulation may be sterilized, for example, by exposure to gamma radiation. In one aspect, the anti-a4ß7 antibody liquid formulation is sterilized by filtration before drying.

[00122] In one aspect, the formulation is storage stable. Various stability tests are available to the practitioner to confirm the stability of the formulation. For example, the antibody in the liquid formulation may be stable in storage at about 25°C for at least about 4 weeks, at least about 2 months, at least about 3 months, or at least about 6 months, or at least about 9 months, or at least about 12 months; at about 2-8oC at least about 3 months, at least about 1 year, at least about 2 years, at least about 3 years or more. Alternatively or in addition, the antibody in the formulation may be stable in storage at about 15°C for at least about 4 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about least about 1 year, or more. Alternatively or in addition, the antibody in the formulation may be stable in storage at about -20°C or -70°C for at least about 4 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years or more.

[00123] Stability can be tested by evaluating the physical stability, chemical stability, and/or biological activity of the antibody in the formulation around the formulation time as well as after storage at the noted temperatures. Physical and/or chemical stability of a liquid formulation or a reconstituted dry powder can be evaluated qualitatively and/or quantitatively in a variety of different ways (see, e.g., Analytical Techniques for Biopharmaceutical Development, Rodriguez-Diaz et al. eds. Informa Healthcare (2005)), including assessment of soluble or insoluble aggregate formation (e.g., using size exclusion chromatography, analytical ultracentrifugation, MALDI-TOF MS, dynamic light scattering ((DLS) or MALLS), microscopic imaging based on flow, or other liquid particle counting system, when measuring turbidity, by density gradient centrifugation and/or by visual inspection); assessing charge heterogeneity using cation exchange chromatography (see also Vlasak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) and Harris et al. J. Chromatogr. B Biomed. Sci. Appl. 752:233 -245 (2001)), isoelectric focusing or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare fragmented, intact, and multimeric (i.e., dimeric, trimeric, etc.) antibody; peptide map (e.g. tryptic or LYS and the like). Instability can result in aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), denaturation, shearing/hydrolysis/fragmentation (e.g., hinge region fragmentation). ), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, glycosylation differences, etc. Biological activity or antigen-binding function, e.g., binding of anti-a4ß7 antibody to MAdCAM (e.g., MAdCAM-1) or inhibition of binding of a cell expressing a4ß7 integrin to MAdCAM (e.g., MAdCAM-1), for example, immobilized MAdCAM (e.g., MAdCAM-1), can be evaluated using various techniques available to the expert practitioner (see, e.g., Soler et al., J. Pharmacol. Exper. Ther. 330:864-875 (2009 )). Measuring the moisture content of a dry formulation can indicate how likely a formulation will undergo chemical or physical degradation, with higher moisture leading to more degradation.

[00124] A stable formulation may contribute to the low immunogenicity of an anti-a4ß7 antibody. An immunogenic anti-a4ß7 antibody can lead to a human-anti-human antibody (HAHA) response in human subjects or patients. Patients who develop a HAHA response to an anti-a4ß7 antibody may experience adverse events (e.g., infusion site reaction) on treatment or may eliminate anti-a4ß7 antibody rapidly, resulting in a lower than planned treatment dose. One report (Feagen et al. (2005) N. Engl. J. Med. 352:2499-2507) of an early study of an anti-a4ß7 antibody treatment indicated that anti-human antibodies developed by week 8 in 44% of treated patients. The antibody in this study was stored as a liquid and did not contain any polysorbate.

[00125] In some embodiments, the formulation can increase the proportion of HAHA-negative patients by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of patients compared to HAHA results from a less stable formulation.

[00126] In some embodiments, an anti-a4ß7 antibody formulation has > 50% charged major isoform, > 55% charged major isoform, or 65 to 70% charged major isoform. In other aspects, a stable anti-a4ß7 antibody formulation has <45% acidic charged isoforms, <40% acidic charged isoforms, <30% acidic charged isoforms, or 22 to 28% acidic isoforms. In still other aspects, a stable anti-a4ß7 antibody formulation has <25% basic isoforms, <20% basic isoforms, <15% basic isoforms, about 5% basic isoforms, or about 10% basic isoforms. . In one aspect, a stable anti-a4ß7 antibody formulation has >55% major isoform, <30% acidic isoforms, and/or <20% basic isoforms, for example, as determined by CEX. In another aspect, a stable anti-a4ß7 antibody formulation has > 50% major isoform, < 45% acidic isoforms and/or < 10% basic isoforms, for example, as determined by cIEF.

[00127] In some aspects, a solid, dry formulation of anti-a4ß7 antibody has <10% moisture content, <5% moisture content, or <2.5% moisture content. The time required for reconstitution is < 60 minutes, < 50 minutes or < 40 minutes or < 30 minutes or < 20 minutes.

[00128] Monomeric content and/or aggregate content (for example, as dimers, trimers, tetramers, pentamers, oligomers and higher order aggregates), i.e. in the liquid formulation, or in the reconstituted formulation, can be measured by SEC, analytical ultracentrifugation, light scattering (DLS or MALLS), MALDI-TOF MS or nanoscale measurement such as NTA nanoparticle tracking analysis, NanoSight Ltd, Wiltshire, UK). Aggregate resolution, characterization and quantification can be achieved in a number of ways, including increasing the length of the SEC column separation, for example, by a larger column or by serial connection of a second or more SEC columns in line with the SEC column. initial analytical, complementary SEC quantification of monomers with light scattering, or using NTA.

[00129] In one embodiment, an anti-a4ß7 antibody formulation has > 90% monomeric antibody, > 95% monomeric antibody, or 97 to 99% monomeric antibody. In another embodiment, most of the material in an anti-a4ß7 antibody formulation has an average radius of < 20 nm, < 15 nm, < 10 nm, or about 5 to about 7 nm. In one aspect, an anti-a4ß7 antibody formulation has >80% lighter heavy chain amount by protein analysis. In one aspect, there is >90% heavy plus light chain. In another aspect, an anti-a4ß7 antibody formulation has < 10% aggregate, < 5% aggregate, < 2.5% aggregate, < 1.5% aggregate, < 1.0% aggregate, or < 0 .5% aggregate. In another aspect, the stable anti-a4ß7 antibody formulation has > 96% monomer and/or < 2.5% aggregate. In yet another aspect, a stable anti-a4ß7 antibody formulation has about 99% monomer and/or about < 1% aggregate.

[00130] Particle sizes, greater than 1 to 2 microns, for example, of aggregates or undissolved excipient, i.e. in the liquid formulation, or in the reconstituted formulation can be measured by obscuration (e.g. particle counting system liquid (HIAC) by Hach Ultra Analytics (Grants Pass, OR)), microscopy, coulter counter, or digital (e.g., flow-based) microscopic imaging-based system such as microfluidic imaging (MFI) by Brightwell (Ottawa, CA) or FLOWCAM® particle imaging analyzer by Fluid Imaging Technologies (Yarmouth, ME). In one aspect, particle size in an anti-a4ß7 antibody preparation is about 30 µm, about 25 µm, about 10 µm, about 5 µm, about 2 µm or 1 µm or less. The amount of particles should be minimized in antibody formulations. In one aspect, an amount of particles in an anti-a4ß7 antibody formulation is < 6000 particles > 10 µm in diameter and/or < 600 particles > 25 µm in diameter in one dose (U.S. Pharmacopoeia Chp. 788, obscuration counting method ; half of those quantities by microscopic quantification method). In another aspect, an amount of particles in a dose of an anti-a4ß7 antibody formulation is about 1000 particles >10 µm and about 0-100 particles >25 µm (MFI method). In yet another aspect, an amount of particles per milliliter, for example, by MFI measurement, in a dose of an anti-a4ß7 antibody formulation is about 500 to about 2000 of 2-10 µm particles per ml, about 50 to about 350 of >10 µm particles per ml and about 0 to about 50 of >25 µm particles per ml. In yet another aspect, an amount of particles in a dose of an anti-a4ß7 antibody formulation is about 500 to about 100,000, about 1,000 to about 5,000, or about 1,500 to about 3,000 of 2-10 µm particles. per ml.

[00131] The viscosity of an anti-a4ß7 antibody formulation can be controlled for subcutaneous or intramuscular administration. Viscosity can be affected by protein concentration and pH. For example, as the protein concentration increases, the viscosity can increase. An increase in pH may reduce the viscosity of the anti-a4ß7 antibody formulation. In some protein formulations, sodium chloride is added to reduce the viscosity of the formulation. Additional components that may affect the viscosity of an anti-a4ß7 antibody formulation are amino acids such as histidine and arginine.

[00132] An anti-a4ß7 antibody formulation can be isotonic (e.g., 250-350 mOsm) or hypertonic (e.g., more than 350 mOsm, more than 450 mOsm, more than 550 mOsm, or more than 650 mOsm), for example, for subcutaneous or intramuscular administration. In one aspect, the anti-a4ß7 antibody formulation is not hypotonic, e.g., less than 250 mOsm. In another aspect, the anti-a4ß7 antibody formulation is about 350 to about 400 mOsm, about 400 to about 450 mOsm, or about 350 to about 450 mOsm.

[00133] Instability leading to denaturation can be assessed by differential scanning calorimetry (DSC). Antibodies have two melting temperatures (Tm) in DSC, for example, Tm1 and Tm2. Certain excipients may affect the stability of the native anti-a4ß7 antibody. A finding of a higher melting temperature when comparing DSC formulations may indicate a more stable anti-a4ß7 antibody formulation with a higher Tm. For example, at pH5.7, the Tm of an anti-a4ß7 antibody formulation is lower, and thus less stable, than at pH 6.5. In one aspect, Tm1 of an anti-a4ß7 antibody formulation is >60oC. In another aspect, the Tm1 of an anti-a4ß7 antibody formulation is about 65°C to about 70°C or about 69°C. In one aspect, Tm2 of an anti-a4ß7 antibody formulation is >80oC. In another aspect, the Tm2 of an anti-a4ß7 antibody formulation is about 82°C to about 88°C or about 86°C.

[00134] In one embodiment, an anti-a4ß7 antibody formulation has a binding affinity or EC50 value of about 60% to about 140% of the reference standard anti-a4ß7 antibody. In one aspect, an anti-a4ß7 antibody in a formulation described herein binds to a4ß7, for example, in a cell (WO98/06248 or US patent 7,147,851), to an extent of about 80% to about 120%. of the reference standard. In another embodiment, an anti-a4ß7 antibody formulation has the ability to inhibit at least 50%, or at least 60% of the binding of a cell expressing a4ß7 integrin to MAdCAM (e.g., MAdCAM-1), e.g. MAdCAM-Ig chimera (see US patent application publication 20070122404, further for reference standard examples).

[00135] As noted above, freezing of the formulation is specifically contemplated here. Thus, the formulation can be tested for freeze-thaw stability. Thus, the antibody in a liquid formulation may be stable upon freezing and thawing of the formulation, for example, the antibody may be stable after one, two, three, four, five or more freeze/thaw cycles.

[00136] In some embodiments, the pharmaceutical formulation is a liquid formulation comprising at least about 60 mg/ml to about 170 mg/ml anti-a4ß7 antibody, a buffering agent (e.g., histidine), and citrate at least about of 5mM. In other embodiments, the formulation is a liquid formulation comprising at least about 60 mg/ml to about 170 mg/ml anti-a4ß7 antibody, a buffering agent (e.g., citrate), amino acid (e.g., arginine), and surfactant. (e.g. polysorbate 80).

[00137] In another embodiment, the formulation comprises at least about 140 mg/ml or about 150 mg/ml to about 170 mg/ml, for example, about 160 mg/ml of an anti-a4ß7 antibody, an buffering agent (e.g., histidine), citrate at least about 5 mM and a free amino acid (e.g., arginine).

[00138] In yet another embodiment, the formulation comprises at least about 160 mg/ml of an anti-a4ß7 antibody, a buffering agent (e.g., histidine), at least about 5 mM citrate, 0.2% polysorbate 80 , and a free amino acid (e.g. arginine). In one embodiment, the buffer concentration in the formulation is about 15 to about 75 mM, about 25 to about 65 mM, or about 50 mM. The free amino acid concentration in the formulation is about 50 to about 250 mM, about 75 to about 200 mM, about 100 to about 150 mM, or about 125 mM; the concentration of polysorbate 80 in the formulation is about 0.05% to 0.4%, about 0.1% to 0.4%, about 0.1% to 0.3%, about 0.1% to 0.25%, about 0.1% to 0.2%, or about 0.2%.

[00139] In some embodiments, the formulation is a solid formulation (e.g., a lyophilized formulation), comprising a mixture of an anti-a4ß7 antibody, citrate, histidine, arginine, polysorbate 80, and a lyoprotectant or a saccharide, such as a non-reducing sugar. The saccharide can be included in the liquid formulation to achieve concentrations of 0% to 20%, or about 6% to about 10%.

[00140] In one embodiment, the formulation is lyophilized and stored as a single dose in a container, e.g., vial, syringe, cartridge, and/or autoinjector. The container can be stored at about 2-8°C or 25°C until administered to a subject in need thereof. The vial may, for example, be a 5, 10 or 20 cm3 vial (e.g. for a dose of 160 mg/ml). The vial may contain at least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg, at least about 540 mg, or at least about 900 mg of anti-a4ß7 antibody. In one aspect, the container contains about 165 mg of anti-a4ß7 antibody.

[00141] In another embodiment, the formulation is liquid and stored as a single dose in one or two vials, cartridges, syringes, or autoinjectors. The vial, cartridge, syringe, or autoinjector can be stored at about 2-8°C until its contents, for example, an anti-a4ß7 antibody, are administered to a subject in need thereof. The vial may, for example, be a 5, 10 or 20 cm3 vial (e.g. for a dose of 160 mg/ml). The vial may contain at least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg, at least about 540 mg, or at least about 900 mg of anti-a4ß7 antibody. In one aspect, the vial contains about 165 mg of anti-a4ß7 antibody. The syringe or cartridge may be a container of 1 mL or 2 mL (e.g., for a dose of 160 mg/mL) or more than 2 mL, e.g., for a larger dose (at least 320 mg or 400 mg or bigger). The syringe or cartridge may contain at least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg, or at least approximately 500 mg of anti-a4ß7 antibody.

[00142] One or more pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington: The Science and Practice of Pharmacy, 21st Edition, Hendrickson, R. Ed. (2005) may be included in the formulation provided as long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include; additional buffering agents; cosolvents; antioxidants including citrate and cysteine; chelating agents such as EDTA; metal complexes (e.g. Zn protein complexes); biodegradable polymers such as polyesters; condoms; container wall lubricants, e.g., silicone, mineral oil, glycerin, or TRIBOGLIDE® (Tribo Film Research, Inc.) derived from perfluoropolyether, to facilitate injection and/or salt-forming counterions such as sodium.

a4ß7 antibodies

[00143] Anti-a4ß7 antibodies suitable for use in the formulations include antibodies from any desired source, such as fully human antibodies, murine antibodies, rabbit antibodies, and the like, and any desired modified antibodies, such as chimeric antibodies, humanized antibodies, and the like. Antigen-binding fragments of any of these types of antibodies, such as Fab, Fv, scFv, Fab' and F(ab')2 fragments, are still suitable for use in the formulations.

[00144] The anti-a4ß7 antibody can bind to an epitope on the a4 chain (e.g., humanized MAb 21.6 (Bendig et al., US Patent 5,840,299)), on the ß7 chain (e.g., FIB504 or a humanized derivative (e.g., Fong et al., US patent 7,528,236)), or to a combinatorial epitope formed by the association of an a4 chain with the ß7 chain. In one aspect, the antibody binds to a combinatorial epitope on the a4ß7 complex, but does not bind to an epitope on the a4 chain or the ß7 chain unless the chains are in association with each other. The association of a4 integrin with ß7 integrin can react to a combinatorial epitope, for example, by bridging the proximity residues present on both chains that together comprise the epitope or by exposure conformationally on one chain, for example, the a4 chain integrin or the ß7 integrin chain, an epitope binding site that is inaccessible to antibody binding in the absence of the appropriate integrin partner or in the absence of integrin activation. In another aspect, the anti-a4ß7 antibody binds to both the a4 integrin chain and the ß7 integrin chain, and thus is specific for the a4ß7 integrin complex. Said antibodies can bind to a4ß7 but do not bind to a4ß1, and/or do not bind to aEß7, for example. In another aspect, the anti-a4ß7 antibody binds to the same or substantially the same epitope as the Act-1 antibody (Lazarovits, A. I. et al., J. Immunol., 133(4): 1857-1862 (1984), Schweighoffer et al., J. Immunol., 151(2): 717-729, 1993; Bednarczyk et al., J. Biol. Chem., 269(11): 8348-8354, 1994). ACT-1 hybridoma cell line, which produces the murine Act-1 monoclonal antibody, was deposited under the provisions of the Budapest Treaty on August 22, 2001, in the name of Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, Mass. 02139, USA, in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., under accession No. PTA-3663. In another aspect, the anti-a4ß7 antibody is a human antibody or a4ß7-binding protein using the CDRs provided in US patent application publication 2010/0254975.

[00145] In one aspect, the anti-a4ß7 antibody inhibits the binding of a4ß7 to one or more of its ligands (e.g., mucosal addressin, e.g., MAdCAM (e.g., MAdCAM-1), fibronectin, and/or or vascular addressin (VCAM)). Primate MAdCAMs (e.g., MAdCAM-1) are described in PCT publication WO 96/24673, the entire teachings of which are incorporated herein by this reference. In another aspect, the anti-a4ß7 antibody inhibits the binding of a4ß7 to MAdCAM (e.g., MAdCAM-1) and/or fibronectin without inhibiting VCAM binding.

[00146] In one aspect, the anti-a4ß7 antibodies for use in the formulations are humanized versions of the mouse Act-1 antibody. Appropriate methods for preparing humanized antibodies are well known in the art. Generally, the humanized anti-a4ß7 antibody will contain a heavy chain that contains the 3 heavy chain complementarity determining regions (CDRs, CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) the mouse Act-1 antibody and appropriate human heavy chain framework regions; and also contains a light chain that contains the 3 light chain CDRs (CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1 antibody and chain structural regions take appropriate human resources. The humanized Act-1 antibody may contain any appropriate human framework region, including consensus framework region, with or without amino acid substitutions. For example, one or more of the structural amino acids may be replaced with another amino acid, such as the amino acid at the corresponding position in the mouse Act-1 antibody. The human constant region or portion thereof, if present, may be derived from the K or À light chains, and/or the y heavy chains (e.g., y1, y2, y3, y4), µ, a (e.g., a1 , a2), d or e of human antibodies, including allelic variants. A particular constant region (e.g., IgG1), variant, or parts thereof can be selected to configure effector function. For example, a mutated constant region (variant) can be incorporated into a fusion protein to minimize binding to Fc receptors and/or ability to fix complement (see, e.g., Winter et al., GB 2,209,757 B; Morrison et al ., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994). Humanized versions of Act-1 antibody have been described in PCT publications WO98/06248 and WO07/61679, the complete teachings of each of which are incorporated herein by this reference.

[00147] In another aspect, the humanized anti-a4ß7 antibodies for use in the formulation comprise a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO:2, and a light chain variable region comprising amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132 of SEQ ID NO:5. If desired, an appropriate human constant region may be present. For example, the humanized anti-a4ß7 antibody may comprise a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:2 and a light chain comprising amino acids 21 to 239 of SEQ ID NO:5. In another example, the humanized anti-a4ß7 antibody may comprise a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:2 and a light chain comprising amino acids 20 to 238 of SEQ ID NO:4. Figure 4 shows an alignment comparing the generic light chains of human antibodies with murine antibodies. The alignment illustrates that the humanized vedolizumab light chain (e.g., Chemical Abstract Service (CAS, American Chemical Society) Registry Number 943609-66-3), with two mouse residues swapped for human residues, is more humane than the LDP-02 light chain (Figure 3). Furthermore, LDP-02 somehow has a hydrophobic, flexible alanine 114 and a hydrophilic (Aspartate 115) site that is replaced in vedolizumab with the slightly hydrophilic hydroxyl-containing threonine 114 residue and hydrophobic valine 115 residue, potentially facing inwards.

[00148] Other substitutions to the antibody sequence may be, for example, mutations to the heavy and light chain structural regions, such as an isoleucine to valine mutation at residue 2 of SEQ ID NO:14; a methionine to valine mutation at residue 4 of SEQ ID NO:14; an alanine to glycine mutation at residue 24 of SEQ ID NO:15; an arginine to lysine mutation at residue 38 of SEQ ID NO:15; an alanine to arginine mutation at residue 40 of SEQ ID NO:15; a methionine to isoleucine mutation at residue 48 of SEQ ID NO:15; an isoleucine to leucine mutation at residue 69 of SEQ ID NO:15; an arginine to valine mutation at residue 71 of SEQ ID NO:15; a threonine to isoleucine mutation at residue 73 of SEQ ID NO:15; or any combination thereof; and replacing the heavy chain CDRs with the CDRs (CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) of the mouse Act-1 antibody; and replacing the light chain CDRs with the light chain CDRs (CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1 antibody.

[00149] In some embodiments, the humanized anti-a4ß7 antibodies for use in the formulation comprise a heavy chain variable region that has about 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 20 to 140 of SEQ ID NO:2, and a light chain variable region that has about 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132 of SEQ ID NO:5. Amino acid sequence identity can be determined using an appropriate sequence alignment algorithm, such as the Lasergene system (DNASTAR, Inc., Madison, Wis.), using default parameters. In one embodiment, the anti-a4ß7 antibody for use in the formulation is vedolizumab (CAS, American Chemical Society, Registration Number 943609-66-3).

[00150] Other a4ß7 antibodies can also be used in the formulations and dosage schedules described here. For example, the a4ß7 antibodies described in US 2010/0254975 (Amgen, Inc.), incorporated by reference herein in their entirety, are suitable for use in formulations and methods for treating inflammatory bowel disease in an individual.

[00151] The anti-a4ß7 antibody can be produced by expressing nucleic acid sequences encoding each chain in living cells, for example, cells in culture. A variety of host expression vector systems can be used to express the antibody molecules of the invention. Host expression systems represent vehicles by which coding sequences of interest can be produced and subsequently purified, but still represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express an anti-a4ß7 antibody. in situ. These include, among others, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid ) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3, NS0 cells) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter; the vaccinia virus 7.5K promoter). For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector as the main human cytomegalovirus intermediate early gene promoter element, is an effective expression system for antibodies (Foecking et al., Gene 45 :101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

[00152] In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when a large amount of said protein must be produced to generate pharmaceutical compositions from an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Said vectors include, among others, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), wherein the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. In general, said fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or Xa protease cleavage sites so that the cloned gene product can be released from the GST moiety.

[00153] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[00154] In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be linked to a transcription/translation control complex, for example, the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may further be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading region of the desired coding sequence to ensure translation of the complete insert. These exogenous translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Expression efficiency can be improved by including appropriate transcription enhancer elements, transcription terminators, etc. (see, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[00155] Additionally, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired. Said modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for primary transcript processing, glycosylation, and phosphorylation of the gene product can be used. Said mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO), NS0, HeLa, VERY, newborn hamster kidney (BHK), monkey kidney (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma cells (e.g. Hep G2), breast cancer cell lines such as BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as CRL7030 and Hs578Bst.

[00156] The glycosylation machine of different cell types can produce antibodies with different glycosylation composition in addition to another cell type, or without glycosylation, as with bacterial cells. In one aspect, the cell types for producing the anti-a4ß7 antibody are mammalian cells, such as NS0 or CHO cells. In one aspect, mammalian cells may further comprise deletion of an enzyme involved in cellular metabolism and the exogenous gene of interest may be operably linked to a replacement enzyme, e.g., in a construct or vector for introduction into cells, e.g. , by transformation or transfection. The construct or vector with the exogenous gene confers on cells hosting the construct or vector a selection advantage to encourage production of the polypeptide encoded by the exogenous gene. In one embodiment, CHO cells are DG44 cells (Chasin and Urlaub (1980) PNAS USA 77:4216), comprising deletion or inactivation of the dihydrofolate reductase gene. In another embodiment, the CHO cells are CHO K1 cells comprising deletion or inactivation of the glutamine synthase gene (see, for example, US patents 5,122,464 or 5,827,739).

Solid formulations

[00157] The solid formulations of the invention are generally prepared by drying a liquid formulation. Any appropriate drying method can be used, such as freeze drying or spray drying. In one aspect, a lyoprotectant is added to the formulation prior to lyophilization. Freeze drying involves freezing a liquid formulation, generally in the container that will be used to store, transport, and dispense a formulation (e.g., a vial, syringe (e.g., a single- or two-chamber syringe), or cartridge (e.g., a single- or two-chamber cartridge) (See, for example, Gatlin and Nail in Protein Purification Process Engineering, ed. Roger G. Harrison, Marcel Dekker Inc., 317-367 (1994).) Once the formulation is frozen, atmospheric pressure is reduced and the temperature is adjusted to allow removal of the frozen solvent, for example, through sublimation. This step in the freeze-drying process is sometimes referred to as a primary drying. If desired, the temperature can then be raised to remove any solvent that is still bound to the dried formulation by evaporation. This step of the freeze-drying process is sometimes referred to as secondary drying. When the formulation has reached the desired degree of dryness, the drying process is completed and the containers are sealed. The final solid formulation is sometimes referred to as a “lyophilized formulation” or a “cake.” The freeze-drying process can be carried out using any appropriate equipment. Appropriate lyophilization equipment is available from a number of commercial sources (e.g., SP Scientific, Stone Ridge, NY).

[00158] A variety of suitable apparatus can be used to dry liquid formulations to produce a solid (e.g., lyophilized) formulation. Generally, lyophilized formulations are prepared by those skilled in the art using a sealed chamber containing shelves, in which vials of the liquid formulation to be dried are placed. The temperature of the shelves, the rate of cooling and heating can be controlled, as can the pressure inside the chamber. It will be understood that several process parameters discussed herein relate to processes carried out using this type of apparatus. Those skilled in the art can easily adapt the parameters described here to other types of drying apparatus if desired.

[00159] Appropriate temperatures and an amount of vacuum for primary and secondary drying can be readily determined by one skilled in the art. In general, the formulation is frozen at a temperature of about -30°C or less, such as -40°C or -50°C. The cooling rate can affect the quantity and size of ice crystals in the matrix. Primary drying is generally conducted at a temperature that is about 10°C, about 20°C, about 30°C, about 40°C or about 50°C but warmer than freezing temperature. In one aspect, the primary drying conditions can be adjusted to maintain the anti-a4ß7 antibody below the glass transition temperature or collapse temperature of the formulation. Above the collapse temperature, the amorphous frozen matrix may flow (collapse), with the result that the protein molecules may not be surrounded by a rigid, solid matrix and the protein molecules may not be stable in the collapsed matrix. Furthermore, the formulation may be difficult to fully dry if collapse occurs. The resulting greater amounts of moisture in the formulation can lead to increased rates of protein degradation and a reduction in the amount of time the freeze-dried product can be stored before its qualities decline to unacceptable levels. In one aspect, the shelf temperature and chamber pressure are selected to maintain the product temperature below the collapse temperature during primary drying. The glass transition temperature of a frozen formulation can be measured by methods known in the art, for example, by differential scanning calorimetry (DSC). The collapse temperature can be measured by methods known in the art, for example, freeze-drying microscopy, optical coherence tomography. The drying step can remove at least 50%, at least 60%, at least 70% or more of the solvent. In one aspect, the primary drying step removes more than 80% of the solvent from the anti-a4ß7 antibody formulation.

[00160] The vial size can be selected based on the surface area that will be exposed to the shelf and freeze-drying during vacuum. Drying time is directly proportional to cake height, thus jar size can be chosen based on what is determined to be a variable cake height. A vial with a large diameter relative to volume can provide a high amount of shelf contact for efficient heat transfer during the freeze drying cycle. An antibody solution diluted in a high volume of liquid will require more time to dry. A balance of bottle size versus formulation volume needs to be struck, because larger bottles can be more expensive to store and transport and have a greater proportion of air space to formulation, and can expose a high proportion of the formulation to the effects of moisture degradation. during long-term storage. For a dose of 165 mg, the vial size of the anti-a4ß7 antibody formulation can be 3 mL, 5 mL, or 10 mL. In one aspect, the vial size is 5 ml for a 160 mg/ml solution.

[00161] The principles for choosing a cartridge or syringe size for freeze drying are similar to those for the vial. The depth of the cake height will further increase the drying time as the height increases. The diameter and size of the syringe or cartridge must be balanced with the volume of the final formulation. Larger diameters can increase the rate of moisture absorption in the freeze-dried cake, thus increasing the effects of moisture degradation during storage. For a dose of 165 mg, the volume of the anti-a4ß7 antibody formulation can be 1 ml or 2 ml. In one aspect, the size of the syringe or cartridge is more than 1 mL for a 160 mg/mL solution.

[00162] After lyophilization, the vial, syringe, or cartridge can be sealed, for example, crimped, in a vacuum. Alternatively, a gas, for example dry air or nitrogen, may be allowed into the container before sealing. Where oxidation is a concern, the gas permitted in the freeze-drying chamber may comprise a gas that retards or prevents oxidation of the freeze-dried product. In one aspect the gases are non-oxygenated gases, for example nitrogen, or an inert gas, for example helium, neon, argon, krypton or xenon. In another aspect, the gas is nitrogen or argon.

Treatment with the antibody formulation

[00163] In one aspect, the invention provides a method for treating a disease or disorder in a subject comprising administering to the subject the anti-a4ß7 antibody formulation described herein in an amount effective to treat the disease or disorder, e.g., in humans. . The human subject may be an adult (e.g., 18 years of age or older), a teenager, or a child. The human subject may be a person 65 years of age or older. In contrast to alternative therapeutic dosage schedules, a human subject 65 years of age or older does not require any modification of the dosage regimen described herein, and may be administered the conventional anti-a4ß7 antibody formulation described herein.

[00164] The subject may have had a lack of an adequate response with, loss of response to, or been intolerant to treatment with an immunomodulator, a TNF-a antagonist, or combinations thereof. The patient may have received prior treatment with at least one corticosteroid (e.g., prednisone) for inflammatory bowel disease. An inadequate response to corticosteroids refers to the signs and symptoms of persistently active disease despite a history of at least a 4-week induction regimen that included a dose equivalent to prednisone 30 mg daily orally for 2 weeks or intravenously for 1 week. A loss of response to corticosteroids refers to two failed attempts to reduce corticosteroids below a dose equivalent to oral prednisone 10 mg daily. Corticosteroid intolerance includes a history of Cushing's syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia and/or infection.

[00165] An immunomodulator can be, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. An inadequate response to an immunomodulator refers to the signs and symptoms of persistently active disease despite a history of at least an 8-week regimen or oral azathioprine (>1.5 mg/kg), 6-mercaptopurine (>0.75 mg/kg), or methotrexate (>12.5 mg/week). Intolerance of an immunomodulator includes, but is not limited to, nausea/vomiting, abdominal pain, pancreatitis, LFT abnormalities, lymphopenia, TPMT genetic mutation, and/or infection.

[00166] A TNF-a antagonist is, for example, an agent that inhibits the biological activity of TNF-a, and preferentially binds to TNF-a, such as a monoclonal antibody, for example, REMICADE (infliximab), HUMIRA (adalimumab ), CIMZIA (certolizumab pegol), SIMPONI (golimumab), or a receptor fusion protein such as ENBREL (etanercept). An inadequate response to a TNF-α antagonist refers to the signs and symptoms of persistently active disease despite a history of at least one 4-week induction regimen of infliximab 5 mg/kg IV, 2 doses at least 2 weeks apart. ; a subcutaneous 80 mg dose of adalimumab, followed by a 40 mg dose at least two weeks apart; or 400 mg of Certolizumab pegol subcutaneously, 2 doses at least 2 weeks apart. A loss of response to a TNF-α antagonist refers to the recurrence of symptoms during maintenance dosing after prior clinical benefit. Intolerance to a TNF-a antagonist includes, but is not limited to, infusion-related reaction, demyelination, congestive heart failure, and/or infection.

[00167] A loss of remission maintenance, as used here for subjects with ulcerative colitis, refers to an increase in Mayo score of at least 3 points and a Modified Baron Score of at least 2.

[00168] In another aspect, the present invention provides anti-a4ß7 antibody formulations that (1) can bind a4ß7 integrin in vitro and/or in vivo; and (2) can modulate an activity or function of an a4ß7 integrin, such as (a) binding function (e.g., the ability of a4ß7 integrin to bind to MAdCAM (e.g., MAdCAM-1), fibronectin and/or VCAM -1) and/or (b) leukocyte infiltration function, including recruitment and/or accumulation of leukocytes in tissues (e.g., the ability to inhibit lymphocyte migration to intestinal mucosal tissue). In one embodiment, an antibody in the formulation can bind an a4ß7 integrin, and can inhibit the binding of a4ß7 integrin to one or more of its ligands (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyte infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues). In another embodiment, an antibody in the formulation can bind to an a4ß7 integrin, and can selectively inhibit the binding of the a4ß7 integrin to one or more of its ligands (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-1, fibronectin ), thereby inhibiting leukocyte infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues). Said anti-a4ß7 antibody formulations can inhibit cellular adhesion of cells containing an a4ß7 integrin to vascular endothelial cells in mucosal tissues, including gut-associated tissues, lymphoid organs or leukocytes (especially lymphocytes such as T or B cells) in vitro and/or in vivo. In yet another embodiment, the anti-a4ß7 antibody formulation of the present invention can inhibit the interaction of a4ß7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. In yet another embodiment, the anti-a4ß7 antibody formulation of the present invention can inhibit the interaction of a4ß7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin selectively, for example, without inhibiting the infection of a4ß7 with VCAM.

[00169] The anti-a4ß7 antibody formulations of the present invention can be used to modulate (e.g., inhibit (reduce or prevent)) the leukocyte (e.g., lymphocyte, monocyte) binding function and/or infiltration function of a4ß7 integrin. For example, humanized immunoglobulins that inhibit the binding of a4ß7 integrin to a ligand (i.e., one or more ligands) can be administered in accordance with the method in the treatment of diseases associated with leukocyte (e.g., lymphocyte, monocyte) infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues), particularly from tissues expressing the MAdCAM molecule (e.g., MAdCAM-1).

[00170] An effective amount of an anti-a4ß7 antibody formulation of the present invention (i.e., one or more) is administered to an individual (e.g., a mammal, such as a human or other primate) to treat said disease. For example, inflammatory diseases, including diseases that are associated with leukocyte infiltration of the gastrointestinal tract (including gut-associated endothelium), other mucosal tissues, or tissues that express the MAdCAM (e.g., MAdCAM-1) molecule (e.g., Gut-associated tissues, such as venules of the lamina propria of the small and large intestine; and mammary gland (e.g., lactating mammary gland), can be treated according to the present method. Similarly, an individual having disease associated with leukocyte infiltration of tissues as a result of leukocyte binding to cells (e.g., endothelial cells) expressing MAdCAM (e.g., MAdCAM-1) may be treated in accordance with the present invention. invention.

[00171] In one embodiment, diseases that can be treated in this manner include inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, celiac disease, non-tropical sprue, enteropathy associated with seronegative arthropathy, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis resulting after protocolectomy, and ileoanal anastomosis. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Ulcerative colitis can be moderately to severely active ulcerative colitis. Treatment may result in mucosal healing in patients suffering from moderately to severely active ulcerative colitis. Treatment may also result in a reduction, elimination or reduction and elimination of corticosteroid use by the patient.

[00172] Pancreatitis and insulin-dependent diabetes mellitus are other diseases that can be treated using the formulations of the invention. It has been reported that MAdCAM (e.g., MAdCAM-1) is expressed by some vessels in the exocrine pancreas of NOD (non-obese diabetic) mice as well as BALB/c and SJL mice. Expression of MAdCAM (e.g., MAdCAM-1) was reported to be induced in endothelium in inflamed islets of the NOD mouse pancreas, and MAdCAM (e.g., MAdCAM-1) was the predominant addressin expressed by NOD islet endothelium at early stages. of insulitis (Hanninen, A., et al., J. Clin. Invest., 92: 25092515 (1993)). Treatment of NOD mice with anti-MAdCAM or anti-ß7 antibodies prevented the development of diabetes (Yang et al., Diabetes, 46:1542-1547 (1997)). Furthermore, accumulation of a4ß7-expressing lymphocytes within islets was observed, and MAdCAM-1 was involved in binding lymphoma cells via a4ß7 to inflamed islet vessels (Hanninen, A., et al., J. Clin. Invest. , 92: 2509-2515 (1993)) or to the gastrointestinal tract in mantle cell lymphoma (Geissmann et al., Am. J. Pathol., 153:1701-1705 (1998)).

[00173] Examples of inflammatory diseases associated with mucosal tissues that can be treated using a formulation of the invention include cholecystitis, cholangitis (Adams and Eksteen Nature Reviews 6:244-251 (2006) Grant et al., Hepatology 33:1065-1072 (2001)), e.g., primary sclerosing cholangitis, Behcet's disease, e.g., of the intestine, or pericolangitis (bile duct and tissue surrounding the liver), and graft-versus-host disease (e.g., in the gastrointestinal tract (e.g. , after a bone marrow transplant) (Petrovic et al. Blood 103:1542-1547 (2004)).As seen in Crohn's disease, inflammation often extends beyond the mucosal surface, thus chronic inflammatory diseases such as sarcoidosis, chronic gastritis, e.g., autoimmune gastritis (Katakai et al., Int. Immunol., 14:167-175 (2002)) and other idiopathic conditions may be amenable to treatment.

[00174] The invention further relates to a method for inhibiting leukocyte infiltration of mucosal tissue. The invention further relates to a method for treating cancer (for example, an a4ß7-positive tumor, such as lymphoma). Other examples of inflammatory diseases associated with mucosal tissues that can be treated using a formulation of the invention include mastitis (mammary gland) and irritable bowel syndrome.

[00175] Diseases or pathogens whose etiologies exploit the interaction of MAdCAM (e.g., MAdCAM-1) with a4ß7 can be treated with an anti-a4ß7 antibody in a formulation described here. Examples of said diseases include immunodeficiency disorders, such as caused by human immunodeficiency viruses (See, for example, WO2008140602).

[00176] A formulation of the invention is administered in an effective amount that inhibits the binding of a4ß7 integrin to a ligand thereof. For therapy, an effective amount will be sufficient to achieve the desired therapeutic (including prophylactic) effect (such as an amount sufficient to reduce or prevent a4ß7 integrin-mediated binding and/or signaling, thereby inhibiting leukocyte adhesion and infiltration and/or responses associated cell phones). An effective amount of an anti-a4ß7 antibody, for example, an effective titer sufficient to maintain saturation, e.g. neutralization, of a4ß7 integrin, can induce clinical response or remission in inflammatory bowel disease. An effective amount of an anti-a4ß7 antibody can lead to mucosal healing in ulcerative colitis or Crohn's disease. A formulation of the invention can be administered in a single dose or multiple doses. The dosage may be determined by methods known in the art and may be dependent, for example, on the individual's age, sensitivity, tolerance and general well-being. Examples of modes of administration include topical routes such as nasal or inhalation or transdermal administration, enteral routes such as by a feeding tube or suppository, and parenteral routes such as intravenous, intramuscular, subcutaneous, intra-arterial, intraperitoneal, or intravitreal administration. Appropriate dosages for antibodies may be from about 0.1 mg/kg body weight to about 10.0 mg/kg body weight per treatment, for example, about 2 mg/kg to about 7 mg/kg, about from 3 mg/kg to about 6 mg/kg, or about 3.5 to about 5 mg/kg. In particular embodiments, the administered dose is about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. The total dose may be about 22 mg, about 50 mg, about 72 mg, about 125 mg, about 165 mg, or about 432 mg. The total dose may be at least 77 mg, at least 125 mg, or at least 356 mg. In one embodiment, the total dose is 165 mg. In another embodiment, the total dose is 108 mg. In another embodiment, the total dose is 216 mg.

[00177] Modeling and simulations (BERKELEY MADONNA™ software, University of California) using pharmacokinetic (PK) data from studies of anti-a4ß7 antibody availability over time after administration can evaluate potential dosing schedules for subcutaneous or intramuscularly. PK data can be evaluated for induction and maintenance regimens. Another modeling approach is population pharmacokinetics/pharmacodynamics analysis (non-linear mixed effects modeling tool NONMEM®, ICON plc, Dublin, Ireland). Both exposure levels and minimum levels can be analyzed.

[00178] Typically, after targeting, for example, a4ß7 integrin, saturation is reached, the concentration of the antibody in the blood has a linear relationship to the dose administered. An anti-a4ß7 antibody administered subcutaneously or intramuscularly has about 60% to about 90% of the bioavailability of an anti-a4ß7 antibody administered intravenously. In an example of this relationship, if an IV dose is assumed to have a bioavailability of 100% and a subcutaneous dose is shown to have a bioavailability of 69.5%, then a 300 mg intravenous dose can be combined with a 432 mg dose per dose. subcutaneous administration. Thus, a 150 mg intravenous dose can be combined with a subcutaneous dose of 216 mg at 69.5% in terms of bioavailability. Similarly, if a subcutaneous dose is shown to have a bioavailability of 75% and an intramuscular dose is shown to have a bioavailability of 80%, then to combine a 300 mg intravenous dose, the subcutaneous dose may be 400 mg and the intramuscular dose may be be 375 mg. Tables 40-43 in the examples illustrate these relationships and provide useful doses and dosing schedules of an anti-a4ß7 antibody.

[00179] In some aspects, the dosage regimen has two phases, an induction phase and a maintenance phase. In the induction phase, the antibody or antigen-binding fragment thereof is administered in a route that rapidly delivers an effective amount of the antibody or antigen-binding fragment thereof appropriate for certain purposes, such as inducing immune tolerance to the antibody or antigen-binding fragment thereof. binding to the same antigen or to induce a clinical response and alleviate the symptoms of inflammatory bowel disease. A patient may be administered an induction phase treatment when first being treated with an anti-a4ß7 antibody, when being treated after a long absence from therapy, e.g., more than three months, more than four months, more than six months, more than nine months, more than one year, more than eighteen months or more than two years after anti-a4ß7 antibody therapy or during the maintenance phase of anti-a4ß7 antibody therapy -a4ß7 if there is a return of symptoms of inflammatory bowel disease, for example, a recurrence of remission of the disease. In some embodiments, the induction phase regimen results in a higher average trough concentration, e.g., the concentration immediately before the next dose, than the average steady-state trough concentration maintained during the maintenance regimen.

[00180] In the maintenance phase, the antibody or antigen-binding fragment thereof is administered in a manner that continues the response obtained by induction therapy with a stable level of antibody or antigen-binding fragment thereof. A maintenance regimen can prevent the return of symptoms or recurrence of inflammatory bowel disease. A maintenance regimen may provide convenience to the patient, for example, be a simple dosing regimen or require infrequent excursions for treatment. In some embodiments, the maintenance regimen may include administration of the anti-a4ß7 antibody or antigen-binding fragment thereof, e.g., in a formulation described herein, by a strategy selected from the group consisting of low dose, infrequent administration, self-administration and any combination of the above.

[00181] In one embodiment, for example, during an induction phase of therapy, the dosage regimen provides an effective amount of an anti-a4ß7 antibody or antigen-binding fragment in a formulation described herein to induce remission of a disease. inflammatory bowel disease in a human patient. In some embodiments, the effective amount of anti-a4ß7 antibody is sufficient to reach about 5 µg/ml to about 60 µg/ml, about 15 µg/ml to about 45 µg/ml, about 20 µg/ml to about 30 µg/ml, or about 25 µg/ml to about 35 µg/ml mean minimum serum concentration of anti-a4ß7 antibody by the end of the induction phase. The duration of the induction phase can be about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks of treatment. In some embodiments, the induction regimen may utilize a strategy selected from the group consisting of high dose, frequent administration, and a combination of high dose and frequent administration of the anti-a4ß7 antibody or antigen-binding fragment thereof, e.g. in a formulation described here. The induction regimen may be one time, or a plurality of more than one dose, for example, at least two doses. During the induction phase, a dose may be administered once a day, every other day, twice a week, once a week, every ten days, once every two weeks, or once every three weeks. In some embodiments, induction doses are administered within the first two weeks of anti-a4ß7 antibody therapy. In one embodiment, the induction regimen may be once at the start of treatment (day 0) and once about two weeks after the start of treatment. In another modality, the duration of the induction phase is six weeks. In another embodiment, the duration of the induction phase is six weeks and a plurality of induction doses are administered during the first two weeks.

[00182] In some embodiments, for example, when initiating treatment of a patient with severe inflammatory bowel disease (e.g., in patients who have failed anti-TNFα therapy), the induction phase needs to be longer in duration than for those patients with severe or moderate disease. In some embodiments, the induction phase for a patient with a serious illness may last at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks. In one embodiment, an induction regimen for a patient with a serious illness may include a dose at week 0 (start of treatment), a dose at week 2, and a dose at week 6. In another embodiment, an induction regimen for a patient with a serious illness may comprise a dose at week 0 (start of treatment), a dose at week 2, a dose at week 6 and a dose at week 10.

[00183] In one embodiment, for example, during a maintenance phase of therapy, the dosage regimen maintains an average minimum steady-state serum concentration, e.g., the plateau concentration immediately before the next dose, of about 5 to about 25 µg/mL, about 7 to about 20 µg/mL, about 5 to about 10 µg/mL, about 10 to about 20 µg/mL, about 15 to about 25 µg/mL or about 9 to about 13 µg/mL of anti-a4ß7 antibody. In another embodiment, the dosage regimen, for example, during a maintenance phase of therapy, maintains a mean steady-state serum concentration of about 20 to about 30 µg/mL, about 20 to about 55 µg/mL, about 20 to about 55 µg/mL, about 20 to about 55 µg/mL, mL, about 30 to about 45 µg/mL, about 45 to about 55 µg/mL or about 35 to about 40 µg/mL of anti-a4ß7 antibody. In another embodiment, the dosage regimen, e.g., during a maintenance phase of therapy, maintains a long-term mean serum concentration, e.g., exposure (e.g., area under the concentration-time curve) of about 15 to about 40 µg/mL, about 10 to about 50 µg/mL, about 18 to about 26 µg/mL, or about 22 to about 33 µg/mL of anti-a4ß7 antibody. In yet another embodiment, the dosage regimen, e.g., during maintenance therapy, maintains a long-term mean serum concentration, e.g., exposure (e.g., area under the concentration-time curve) of about 35 to about 90 µg/mL, about 45 to about 75 µg/mL, about 52 to about 60 µg/mL or about 50 to about 65 µg/mL of anti-a4ß7 antibody.

[00184] The final dosage form may comprise the entire dose in about 0.5 ml, in about 1 ml, in about 1.5 ml, in about 2 ml, in about 2.5 ml, in about of 3 ml of the antibody formulation.

[00185] The final dosage form for intravenous administration may be at a concentration of between about 1.0 mg/ml to about 1.4 mg/ml, about 1.0 mg/ml to about 1.3 mg/ml, about 1.0 mg/ml to about 1.2 mg/ml, about 1.0 to about 1.1 mg/ml, about 1.1 mg/ml to about 1, 4 mg/ml, about 1.1 mg/ml to about 1.3 mg/ml, about 1.1 mg/ml to about 1.2 mg/ml, about 1.2 mg/ml to about 1.4 mg/ml, about 1.2 mg/ml to about 1.3 mg/ml, or about 1.3 mg/ml to about 1.4 mg/ml. The final dosage form may be in a concentration of about 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.8 mg/ml or about 2.0 mg/ml.

[00186] The dose can be administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 10 weeks. A larger or more frequent dose, e.g., every other day, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks may be helpful in inducing remission of active disease. or to treat a new patient, for example, to induce tolerance to anti-a4ß7 antibody. One dose once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 10 weeks , may be useful for preventive therapy, for example, to maintain remission in a patient with a chronic disease. In one aspect, the treatment regimen is treatment on day 0, about week 2, about week 6, and every 1 or 2 weeks thereafter. In another aspect, the treatment regimen for induction is treatment every other day for a total of 6 treatments.

[00187] The dosage regimen can be optimized to induce a clinical response and clinical remission in the patient's inflammatory bowel disease. In some embodiments, the dosage regimen does not alter the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving treatment.

[00188] In some aspects, a durable clinical remission, for example, a clinical remission that is sustained for at least two, at least three, at least four visits with a treating physician within a period of six months or one year after onset of treatment, can be achieved with an optimized dosage regimen.

[00189] In some aspects, a durable clinical response, for example, a clinical response that is sustained for at least 6 months, at least 9 months, at least one year, after initiation of treatment, can be obtained with a regimen of Optimized dosage.

[00190] The formulation can be administered subcutaneously in single or multiple injections. For example, the volume of a single injection can vary from about 0.5 ml to about 3 ml. In one embodiment, the volume of a single injection may be about 0.6 ml to about 1.1 ml or about 1 ml to about 3 ml. In one aspect, the volume of a single injection is about 1 ml. The needle gauge used to administer the formulation subcutaneously may be about 25, about 26, about 27, about 28, about 29 or about 30G.

[00191] The formulation can be administered intramuscularly in single or multiple injections. For example, the volume of a single injection may vary from about 0.5 ml to about 5 ml. In one embodiment, the volume of a single injection may be about 2 ml to about 5 ml, about 0.6 ml to about 1.1 ml, or about 1 ml to about 3 ml. In one aspect, the volume of a single injection is about 1 ml, about 2 ml, about 3 ml, about 4 ml, or about 5 ml. The needle used to administer the formulation intramuscularly may be about 5/8", about 7/8", about 1", about 1.25", about 1.5", about 2", or about 3”. The needle gauge can be between 20-22G for intramuscular administration.

[00192] In one aspect, the invention relates to a method for treating a human patient suffering from inflammatory bowel disease, wherein the method comprises the step of administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the following dosage regimen: (a) initial doses of 165 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every other day for six doses; (b) followed by a seventh and subsequent 165 mg doses of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every two weeks or every four weeks as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[00193] In one aspect, the invention relates to a method for treating a human patient suffering from inflammatory bowel disease, wherein the method comprises the step of administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin, wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the following dosage regimen: (a) an initial intravenous dose of 300 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a intravenous infusion; (b) followed by a subsequent second intravenous dose of 300 mg of a humanized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion about two weeks after the initial dose; (c) followed beginning in week six by a third and subsequent doses of 165 mg of a humanized immunoglobulin or antigen-binding fragment thereof as a subcutaneous injection every week, every two weeks, or every three weeks as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[00194] In another aspect, the invention relates to a dosage regimen for the therapeutic treatment of inflammatory bowel disease, wherein the dosage regimen comprises the step of: administering to a patient suffering from inflammatory bowel disease, an immunoglobulin humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of non-human origin humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to a subcutaneous or intramuscular dosage regimen that maintains a mean steady-state trough serum concentration of about 9 to about 13 µg/ mL of the antibody or antigen-binding fragment thereof; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

[00195] In another aspect, the invention relates to a dosage regimen for the therapeutic treatment of inflammatory bowel disease, wherein the dosage regimen comprises the step of: administering to a patient suffering from inflammatory bowel disease, an immunoglobulin humanized immunoglobulin or antigen-binding fragment thereof containing binding specificity for human a4ß7 integrin, wherein the humanized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of non-human origin human, wherein the humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to a subcutaneous or intramuscular dosing regimen that maintains mean steady-state serum trough concentrations of about 35 to about 40 µg/mL the antibody or antigen-binding fragment thereof; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the a4ß7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDR1, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:11; heavy chain: CDR1 SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3 SEQ ID NO:14.

[00196] In some embodiments, the treatment method, dose or dosage regimen reduces the likelihood that a patient will develop a HAHA response to the anti-a4ß7 antibody. The development of HAHA, for example, as measured by antibodies reactive to the anti-a4ß7 antibody, may increase the clearance of the anti-a4ß7 antibody, e.g., reduce the serum concentration of the anti-a4ß7 antibody, e.g., reduce the number of antibodies anti-a4ß7 linked to a4ß7 integrin, thus making the treatment less effective. In some embodiments, to prevent HAHA, the patient may be treated with an induction regimen followed by a maintenance regimen. In some embodiments, there is no break between the induction regime and the maintenance regime. In some embodiments, the induction regimen comprises administering a plurality of doses of anti-a4ß7 antibody to the patient. To prevent HAHA, the patient can be treated with a high initial dose, e.g., at least 1.5 mg/kg, at least 2 mg/kg, at least 2.5 mg/kg, at least 3 mg/kg, at least 5 mg/kg, at least 8 mg/kg, at least 10 mg/kg, or about 2 to about 6 mg/kg, or frequent initial administrations, e.g., about once a week, about once once every two weeks or about every three weeks, the standard dose when initiating therapy with an anti-a4ß7 antibody. In some embodiments, the treatment method maintains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of patients. like HAHA-negative. In other embodiments, the treatment method maintains patients as HAHA-negative for at least 6 weeks, at least 10 weeks, at least 15 weeks, at least six months, at least 1 year, at least 2 years, or for the duration of therapy. In some embodiments, patients, or at least 30%, at least 40%, at least 50%, or at least 60% of patients who developed HAHA maintain a low titer, e.g., <125, of anti-a4ß7 antibody. In one embodiment, the treatment method maintains at least 70% of patients as HAHA-negative for at least 12 weeks after initiating therapy with an anti-a4ß7 antibody.

[00197] The formulation can be administered to an individual (e.g., a human) alone or in conjunction with another agent. A formulation of the invention may be administered prior to, along with, or subsequent to administration of the additional agent. In one embodiment, more than one formulation that inhibits the binding of a4ß7 integrin to its ligand is administered. In said embodiment, an agent, for example, a monoclonal antibody, such as an anti-MAdCAM or an anti-VCAM-1 monoclonal antibody can be administered. In another embodiment, the additional agent inhibits the binding of leukocytes to an endothelial ligand in a pathway other than the a4ß7 pathway. Said agent may inhibit the binding of, for example, lymphocytes expressing chemokine (C-C motif) receptor 9 (CCR9) to the thymus-expressed chemokine (TECK or CCL25) or an agent that prevents the binding of LFA-1 to intercellular adhesion molecule (ICAM). For example, an anti-TECK or anti-CCR9 antibody or a small molecule CCR9 inhibitor, such as inhibitors disclosed in PCT publication WO03/099773 or WO04/046092, or anti-ICAM-1 antibody or an oligonucleotide that prevents ICAM expression , is administered in addition to a formulation of the present invention. In yet another embodiment, an additional active ingredient (e.g., an anti-inflammatory compound such as sulfasalazine, azathioprine, 6-mercaptopurine, 5-aminosalicylic acid containing anti-inflammatory compounds, another non-steroidal anti-inflammatory compound, an anti-inflammatory compound steroidal agent, or antibiotics commonly administered to control IBD (e.g., ciprofloxacin, metronidazole), or other biological agent (e.g., TNF alpha antagonists) may be administered in conjunction with a formulation of the present invention.

[00198] In one embodiment, the dose of co-administered medication can be reduced over time during the treatment period by a formulation comprising an anti-a4ß7 antibody. For example, a patient being treated with a steroid (e.g., prednisone, prednisolone) at the beginning of, or prior to, treatment with the anti-a4ß7 antibody formulation undergoes a regimen of decreasing doses of steroid starting as early as 6 weeks of treatment with the anti-a4ß7 antibody formulation. The steroid dose will be reduced by approximately 25% within 4-8 weeks of tapering, by 50% within approximately 8-12 weeks, and 75% within approximately 12-16 weeks of tapering during treatment with the formulation. of the anti-a4ß7 antibody. In one aspect, in about 1624 weeks of treatment with the anti-a4ß7 antibody formulation, the steroid dose can be eliminated. In another example, a patient being treated with an anti-inflammatory compound such as 6-mercaptopurine at the beginning of, or prior to, treatment with the anti-a4ß7 antibody formulation could undergo a regimen of reduced doses of a similar anti-inflammatory compound. to the tapering regimen for steroid dosage as noted above.

[00199] In one embodiment, the method comprises administering subcutaneously or intramuscularly administering an effective amount of a formulation of the invention to a patient. In another embodiment, the formulation can be prepared for self-administration.

[00200] If the formulation is in a solid state, for example, dry, the administration process may comprise a step of converting a formulation to a liquid state. In one aspect, a dry formulation may be reconstituted, for example, by a liquid as described above, for use in injection, e.g., intravenous, intramuscular or subcutaneous injection. In another aspect, a solid or dry formulation can be administered topically, for example, in a patch, cream, aerosol or suppository.

[00201] The invention further relates to a method for treating a disease associated with leukocyte infiltration of tissues expressing the MAdCAM molecule (e.g., MAdCAM-1). The method comprises administering to a patient in need thereof an effective amount of an anti-a4ß7 antibody formulation of the invention. In one embodiment, the disease is graft versus host disease. In some embodiments, the disease is a disease associated with leukocyte infiltration of tissues as a result of binding of leukocytes expressing a4ß7 integrin to the gut-associated endothelium expressing the MAdCAM molecule (e.g., MAdCAM-1). In other embodiments, the disease is gastritis (e.g., eosinophilic gastritis or autoimmune gastritis), pancreatitis, or insulin-dependent diabetes mellitus. In still other modalities, the disease is cholecystitis, cholangitis, or pericolangitis.

[00202] The invention further relates to a method for treating inflammatory bowel disease in a patient. In one embodiment, the method comprises subcutaneously administering to the patient an effective amount of an anti-a4ß7 antibody formulation of the invention. In some embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In other embodiments, the inflammatory bowel disease is celiac disease, enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, gastroenteritis (e.g., eosinophilic gastroenteritis), or pouchitis.

[00203] In some embodiments, treatment with an anti-a4ß7 antibody does not alter the ratio of CD4:CD8 lymphocytes. CD4:CD8 ratios can be measured in blood, lymph node aspirate, and cerebrospinal fluid (CSF). The ratios of CSF CD4+:CD8+ lymphocytes in healthy individuals are typically greater than or equal to about 1. (Svenningsson et al., J. Neuroimmunol. 1995;63:39-46; Svenningsson et al., Ann Neurol. 1993 ; 34:155-161). An immunomodulator can change the CD4:CD8 ratio to less than 1.

Crafting Items

[00204] In another aspect, the invention is an article of manufacture that contains the pharmaceutical formulation of the present invention and provides instructions for its use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g., two-chamber vials, a vial of liquid formulation with or without a needle, a vial of solid formulation with or without a vial of reconstitution liquid with or without a needle) , syringes (such as two-chamber syringes, prefilled syringes, an autoinjector), cartridges, and test tubes. The container can be formed from a variety of materials such as glass, metal or plastic. The container holds the formulation and a label on, or associated with, the container may indicate instructions for use. In another embodiment, the formulation may be prepared for self-administration and/or contain instructions for self-administration. In one aspect, the container holding the formulation may be a single-use vial. In another aspect, the container holding the formulation may be a multipurpose vial, which allows repeated administration (e.g., 26 administrations) of the formulation, for example, using more than one portion of a reconstituted formulation. The article of manufacture may also include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes and leaflets with instructions for use as noted in the section above.

[00205] In one embodiment, the article of manufacture is a syringe with a needle. The needle gauge may be 25 G, 26 G, 27 G, 29 G, 30 G. A thin-walled needle, for example, 19 G or 23 G, or larger, may facilitate injection of a high viscosity formulation. In one aspect, the needle gauge is 27 G or more. The length of the needle may be appropriate for subcutaneous administration, and may be about 1/2 inch, about 5/8 inch or 1 inch in length. In some embodiments, the syringe is a prefilled syringe.

Prefilled syringe product development

[00206] In some aspects, there are various product attributes that are desired for a protein product (e.g., an anti-a4ß7 antibody) in a pre-filled syringe (PFS) (e.g., for use in administering a formulation for release subcutaneous or intramuscular). It is also useful to balance some of the attributes to mitigate the effects of competition. For example, when a low injection volume is desired, a high protein concentration for the formulation may be preferred. However, in the case of a high protein concentration, there may be higher rates of impurity formation (e.g., aggregated impurities leaking into the syringe formulation) and greater manual forces required to operate the syringe. A needle that is too small for patient comfort at the injection site may require more force to operate the syringe. An understanding of how product stability and performance are affected by both formulation and syringe parameters such as protein concentration, pH, and needle internal diameter aids the development of a protein product (e.g., an anti-a4ß7 antibody in a prefilled syringe).

[00207] In one aspect, a method for developing a protein product (e.g., an anti-a4ß7 antibody) for use in a prefilled syringe comprises varying syringe parameters and formulation parameters together, e.g., in a coordinated fashion or simultaneously. This can lead to a better understanding of the range of protein stability and product performance that can be expected from a protein product in a prefilled syringe than if each aspect is varied separately or in series.

[00208] The development of a prefilled syringe product (e.g., an anti-a4ß7 antibody) refers to the understanding that at some point, there is a liquid formulation in contact with various components of the prefilled syringe (Figure 15). For example, the formulation may be in contact with a syringe barrel, which may be constructed of glass (e.g., type I borosilicate glass) or plastic (e.g., cyclic olefin polymer (COP), cyclic olefin copolymer (COP). COC), polypropylene or polytetrafluoroethylene). The formulation may be in contact with the syringe, plunger and/or tip cap, which may be elastomeric (e.g., of the same or different materials (e.g., plastic, such as polyethylene, polystyrene, or polypropylene) or elastic, such as rubber (natural , synthetic, butyl) or silicone)). The formulation may be in contact with a lubricant that is added to an internal surface of the cylinder to facilitate piston movement. The lubricant can be, for example, silicone oil, mineral oil or glycerin. In the bounded needle syringe embodiment, there may be a metal alloy needle (e.g., stainless steel needle and adhesive used to collect the needle in place). One consideration for a protein product in a prefilled syringe is that the liquid protein solution is in direct contact with one or more of these components of the syringe throughout the useful life of the product. Both formulation and syringe components can have an impact on product stability.

[00209] Formulation parameters that may affect the stability of the prefilled syringe product include protein concentration, pH, buffer type, buffer concentration, ionic strength, stabilizer type, and stabilizer concentration. Examples of stabilizers for protein formulations include, for example, ionic salts, polysaccharides, amino acids, antioxidants, chelators, and surfactants as described in the previous sections.

[00210] Syringe components that may affect the stability of the prefilled syringe product include, for example, lubricant, plunger and tip cap composition, and impurities. The amount of lubricant (e.g. silicone oil in the syringe barrel) may affect the stability of the product. The plunger and tip cap composition, which can affect the oxygen permeability of these components and introduces leachables from these components into the protein product (e.g., the anti-a4ß7 antibody formulation) can further affect the stability of the product. Another parameter that may affect product stability includes the type and/or amount of impurities (e.g., heavy metal (e.g., tungsten)) that may leach into the product formulation (e.g., from the cylinder (e.g., cylinder glass) and/or needle (e.g. stainless steel needle)). (see also Ludwig et al. J. Pharm. Sci. 99:1721-1733 (2010); Nashed-Samuel et al., American Pharmaceutical Review Jan/Feb:74-80 (2011); Badkar et al. AAPS PharmSciTech 12 :564-572)

[00211] A prefilled syringe can be injected manually or used with an autoinjector device. Functional testing of the prefilled syringe includes measuring the release force, the force required to initiate the plunger movement, and the sliding force, the force required to inject the contents of the syringe at a constant rate. The mechanical performance of the prefilled syringe may be dependent on various formulations and syringe parameters such as the viscosity of the formulation and the amount of lubricant (e.g., silicone oil) in the syringe.

[00212] Various attributes of protein products in prefilled syringes and formulation or syringe factors that may impact those product attributes are shown in Table 1. Many product attributes may be a complex function of various formulation and syringe parameters. For example, syringe sliding force is a function of formulation viscosity, although viscosity can be dependent on several formulation factors, such as protein concentration, stabilizer concentrations, and pH. Table 1: Product Attributes for Protein Products in Prefilled Syringes and the Potential Formulation and Syringe Parameters that May Impact These Attributes

[00213] In one aspect, a surfactant such as polysorbate 20 or polysorbate 80 can be added to protein formulations in prefilled syringes (e.g., to prevent protein molecules from adsorbing or denaturing at liquid/air and/or liquid interfaces). /lubricant (e.g. silicone oil)). Surface adsorption and denaturation of protein molecules may be a mechanism for the nucleation of visible and subvisible protein particles. The addition of a surfactant to a prefilled syringe, therefore, can reduce the formation of visible and subvisible particles in prefilled syringe products. In one embodiment, a small amount of surfactant can emulsify lubricant (e.g., silicone oil droplets in the solution and thus the formation of subvisible and visible lubricant (e.g., silicone oil droplets)) (Ludwig et al. , above). In another embodiment, the amount of surfactant in a formulation is minimized, due to the potential harmful effects of large amounts in protein formulations. Peroxide impurities in polysorbates can lead to increased protein oxidation (Wang and Wang J. Pharm. Sci. 91:2252-2264 (2002)). Large amounts of surfactant can emulsify a significant amount of silicone oil from the syringe walls and leads to an increase in functional glide force over service life. Product development studies can be designed to evaluate the effect of varying surfactant levels on both product stability and syringe performance.

[00214] The complex interactions between formulation and syringe parameters in protein/PFS systems are amenable to evaluating these systems using a Quality by Design (QbD) or Design of Experiments (DOE) approach. Studies can be designed that simultaneously vary formulation and syringe parameters to gain a better understanding of these complex systems. This results in a comprehensive approach to prefilled syringe product development. Table 2 shows an example of the input parameters and levels that may pass through an experimental design for a prefilled syringe product and an example of the analytical test to be employed. Depending on the type of experimental design that is used for the QbD study, the number of experiments could vary from 9 for a screening design to 81 for a full factorial design (all possible combinations). The greater the number of experiments, the greater the number of interactions between product parameters that can be resolved. Software designed for this analysis, e.g., JMP® Statistical Discovery Software (Cary, NC), may be useful for QbD studies. This analysis results in a quantitative understanding of how formulation and syringe parameters interact to impact product attributes. Table 2: Example of an Experimental Design for a Liquid Protein Product in a Prefilled Syringe

[00215] An example of a predictive model that can be obtained from the example experiment shown in Table 2 is shown below, where Cn are numerical constants.

[00216] Soluble aggregate formation over time = C0 + C1 [Protein concentration] + C2 [Protein concentration]2 + C3 [pH] + C4 [Surfactant concentration] + C5 [amount of lubricant]

[00217] Various syringe parameters can affect product stability and performance, therefore, one embodiment includes characterizing how allowable tolerances in syringe parameters affect product stability and performance. The amount of lubricant (e.g. silicone oil) in the syringe barrel can vary 50-100% from syringe to syringe. This variation in quantity can affect various product characteristics as shown in Table 1. The inner diameter of the syringe barrel can vary from syringe to syringe which affects injection forces. For delimited (fixed) needle syringes, the internal diameter may vary from batch to batch or manufacturer to manufacturer, which will affect injection forces. By using a QbD approach to evaluate how syringe parameters affect performance, predictive models can be obtained that can be used to estimate how allowable tolerances in syringe parameters can affect product performance. The predictive models that are obtained using a QbD approach can be used to select formulation and syringe parameters that achieve desired product attributes and to predict product stability and performance.

[00218] The prefilled syringe may contain an addition of silicone or tungsten emulsion to the protein formulation. Exemplary amounts of silicone that may be present in the prefilled syringe range from about 0.3 mg to about 0.8 mg. In one aspect, the amount of silicone that may be present in the prefilled syringe is about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg , or about 0.8 mg. In another aspect, the viscosity of the formulation will vary from 2 to 60 cP, resulting in injection forces of 5N to 80N at a speed of 200 mm/min. In yet another aspect, the viscosity of the formulation will vary from 4 to 27 cP resulting in injection forces of 10 N to 40 N at a speed of 200 mm/min.

[00219] The invention will be better understood by reference to the following examples. It should not, however, be construed as limiting the scope of the invention. All literature and patent citations are incorporated herein by reference.

PROTOCOL FOR PREPARING THE FORMULATION

[00220] An anti-a4ß7 antibody solution is diafiltered in a tangential flow filtration system to reach a specified concentration in citrate, histidine, arginine buffer, then pooled and mixed with a solution of polysorbate 80 in citrate, histidine, arginine buffer . The solution is stored at -70°C in 2L or 5L bottles. The solution is then thawed and filtered twice through a 0.2 µm filter. Approximately 1.0 mL is filled into a sterilized syringe and closed with a sterile plunger (stopper). The formulation is stored and the final drug product is transported in syringes at 2-8°C.

EXAMPLES EXAMPLE 1 MANUFACTURE OF THE FORMULATION Factors Excipient Concentrations

[00221] The formation of aggregates in the antibody formulation was tested. A model of SEC aggregates was developed from experimental data that evaluated protein concentration, pH, and surfactant:protein molar ratio. In a pH range of 6.0 to 6.5, aggregate formation was similar with polysorbate 80 at a protein molar ratio of 0.7 to 1.5. (Fig 6). Generally, at PS80:protein ratios greater than 1.5 the rate of aggregate formation increases with increasing pH. (Fig 7).

[00222] An experiment evaluating the formation of SEC aggregates in the presence of air was carried out. Eleven different variant composition formulations were placed in borosilicate vials and capped with elastomeric stoppers with an air gap. An identical set of formulations was created, and the air space was displaced with argon. These samples were placed in stability at 40°C for two weeks. All samples with air space resulted in large amounts of aggregates at the end of the experiment compared to the same formulation with argon air space. Table 3

[00223] Based on these experiments, it was hypothesized that SEC aggregates form through oxidation or disulfide bond formation. The addition of antioxidants and/or chelators has been explored. A formulation containing 40 mM Histidine, 90 mM Arginine, and 160 mg/mL protein with a molar ratio of 80 polysorbate to protein of 1.5 at pH 6.6 was prepared. To the formulation, 25 mM citrate, 5 mM citrate, 5 mM EDTA, 25 mM cysteine, or 5 mM cysteine were added. All 3 additional excipients reduced aggregate formation (Fig 8). The addition of antioxidants and/or chelators were ranked in order of performance as citrate>EDTA>cysteine. Either 5 or 25 mM citrate reduced the formation of SEC aggregates compared to the control formulation.

[00224] An experiment was carried out to determine the effects of pH, protein concentration, citrate concentration, histidine concentration and the molar ratio of polysorbate 80 to protein. pH was varied from 6.0 to 6.3, protein concentration was varied from 60 to 160 mg/mL, citrate concentration was varied from 0 to 25 mM, histidine concentration was varied from 25 to 50 mM , and the molar ratio of polysorbate 80 to protein was varied from 0.7 to 1.5. The formulations were filled in 1 ml syringes, 27G1/2” (0.55 +/- 0.2 mg silicone). All formulations contained approximately 125 mM arginine.

[00225] Stability was tested at 40°C for two weeks, using CEX and SEC. The results (Fig. 9 and Table 4) show a reduction in aggregate formation with the presence of 25 mM citrate in the formulation, while increasing the protein concentration increased the rate of aggregate formation. The amount of monomer shows opposite trends to aggregate formation at 25°C and 40°C, while at 5°C the amount of monomer is essentially unchanged for up to 24 months (Table 5).

[00226] Another set of formulations explored the rate of formation of SEC aggregates in the presence of 40-63 mM citrate but without histidine at 40°C, 25°C, 5°C. The rate of aggregate formation in these formulations was slightly higher than the histidine formulations at 40°C. However, at 5°C, the rate of aggregate formation in the formulations with citrate and without histidine were comparable to the formulations containing citrate and histidine (Table 6). Furthermore, at 5°C, the amount of monomer is essentially unchanged for up to 24 months (Table 7). pH

[00227] Several pH experiments were performed to determine the effects of pH on CEX degradation at 5°C. The vedolizumab antibody formulation comprised 160 mg/ml anti-a4ß7 antibody, 125 mM arginine, 50 mM histidine, and 25 mM citrate. Several different pH levels, 6.3, 6.5, 6.7 and 6.9 were tested for stability at 40°C, 25°C and 5°C.

[00228] CEX models at 40°C show (FIG. 10) that pH influences CEX degradation for the most part. The pH of formulations containing histidine reduces with increasing temperature, however, the pH of citrate formulations has been shown to not be affected by temperature (FIG. 11). The histidine/citrate formulation was determined to have good stability at a pH of 6.8 at 40°C after 1 week, 6.3-6.5 at 25°C after 6 months, and 6.3-6.5 at 5°C after 6 months. Based on additional studies, formulation stability was similar at 25°C and 5°C for a pH range of 6.2 to 6.9 (Tables 8 and 9).

EXAMPLE 2 STABILITY

[00229] Four different anti-a4ß7 antibody formulations were tested for stability over the course of twelve months. Formulations containing a pH of 6.0-6.2 demonstrated approximately 1-2% fewer major species than formulations containing a pH of 6.3-6.4 (FIG. 12). Formulations containing a pH of 6.3-6.4 showed less than 1% change in basic or major species at 5°C.

[00230] Ten different anti-a4ß7 antibody formulations were tested for stability by SEC over the course of twelve months (Table 10). Formulations with 60 mg/mL protein concentration and containing 25 mM citrate had a change in aggregates of 0.10.2% after 1 year, while formulations containing 160 mg/mL protein and 25 mM citrate had an increase in aggregates. from 0.2-0.3% for 1 year. There was a 0.4-0.6% increase in aggregates for formulations containing 60, 110, or 160 mg/mL of citrate-free protein. Table 10

EXAMPLE 3 VISCOSITY

[00231] The injection force required to administer the pharmaceutical formulation is related to the viscosity of the formulation. Formulations with varying pH and varying concentrations of protein, arginine, histidine, citrate, sucrose, and polysorbate 80 were made. The viscosity of these formulations was tested. A statistical Ln (viscosity) model was developed. The model showed that viscosity is mainly affected by protein concentration and pH (Fig. 13). Sucrose, histidine and arginine may still have a minor effect on viscosity. In some protein formulations, sodium chloride is added to reduce the viscosity of the formulation. It is known, however, that the effect of sodium chloride on viscosity is protein and formulation dependent.

[00232] Sodium chloride was added to the formulation containing 140 mg/ml vedolizumab, 125 mM arginine, 25 mM histidine, 25 mM citrate, and polysorbate 80 at a molar ratio of 1.5 polysorbate 80 to protein, and a pH of 6 ,4. NaCl had no effect on the viscosity of the formulation.

[00233] The effects of viscosity on the injection force of various syringes tested are shown in Figures 16A and 16B.

EXAMPLE 4 METHODS Cation exchange chromatography (CEX)

[00234] A phosphate/sodium chloride gradient on a weak cation exchange column is used in a high-performance liquid chromatography system to separate charged species in anti-a4ß7 antibody formulations and determine the charge composition of the antibody species . Acidic isoforms elute before the main isoform and basic isoforms elute after the main isoform.

[00235] Stability data for a vedolizumab formulation generated using a CEX assay indicated that the % major isoform was above 55.0%.

Capillary isoelectric focusing (cIEF)

[00236] cIEF is performed using an iCE280 complete column in cIEF detection system (Convergent Biosciences, Toronto, Ontario). The choice of ampholyte may be recommended by the manufacturer or may be a combination of commercially available ampholytes. A useful combination is a mixture of 3-10 and 5-8 PHARMALYTE™ (GE Healthcare, Piscataway, NJ).

[00237] Stability data for a vedolizumab formulation generated using a cIEF assay indicated that the % major isoform was about 53%, the % acidic species was about 42%, and the % basic species was about 5 %.

Size exclusion chromatography (SEC)

[00238] SEC is performed using an SEC analytical column (Tosoh Bioscience, LLC, King of Prussia, PA). The mobile phase is a phosphate buffered saline solution and the absorbance is monitored at 280 nm.

[00239] Stability data for a vedolizumab formulation generated using an SEC assay indicated that the % monomer was 99.0%, the % aggregates was <0.5%, and the % low molecular weight substances was < 1.0%.

SDS-PAGE Assay

[00240] SDS-PAGE is performed using an Invitrogen (Carlsbad, CA) Tris-Glycine gel, 4-20% for reducing condition and 4-12% for non-reducing condition. The reconstituted antibody formulation is diluted in liquid formulation buffer, then diluted one to two with Tris-Glycine SDS sample buffer (Sample 2X, Invitrogen) with 10% 2-mercaptoethanol (reducing sample buffer) or without 2- mercaptoethanol (non-reducing sample buffer). Samples are briefly heated and loaded against a high molecular weight tracer (Invitrogen). Gels are stained with colloidal coomassie blue (Invitrogen) according to the manufacturer's instructions. Protein bands are analyzed by densitometry to identify % heavy chain and light chain and % IgG for non-reduced gels.

Binding Effectiveness

[00241] HuT78 cells (human T-cell lymphoma cells, American Type Culture Collection, Manassas, VA) suspended in 1% BSA in PBS, 0.01% sodium azide are contacted with serial dilutions of primary test antibody. After incubation on ice, cells are washed and treated with fluorescently labeled secondary antibody. After another wash, the cells are fixed and suspended in FACS reagent for analysis by flow cytometry (Becton Dickinson Franklin Lakes, NJ); see also US patent 7,147,851. Humidity by Karl Fischer

[00242] The formulation is titrated with methanol for a Karl Fischer coulometric humidity determination.

EXAMPLE 5 Effects of Silicone Products in Syringe Filled with Anti-a4ß7 Antibody Formulation

[00243] A subcutaneous formulation consisting of 60-160 mg/mL of anti-a4ß7 protein in a buffer containing L-Histidine, L-Arginine Hydrochloride, Citrate and Polysorbate 80 is used to study the effects of silicone on the stability of formulations of protein and container/closure attributes. The study is carried out with a 0.5 mL bottle.

[00244] Parameters including protein concentration, the molar ratio of polysorbate 80 to protein, and the amount of silicone that is sprayed into the syringe barrels are explored. The range of each of the input parameters is shown in Table 11. Table 11. Input parameter ranges

[00245] An experimental design is used to determine the set of formulations for the study. A reasonable number of formulations ranges from 6 to 8 formulations. An example of formulations that are tested is shown in Table 12. Table 12

[00246] Some controls can be added to the set of formulations and tested at a few selective time points.

[00247] These formulations are set to stability at various different temperatures (e.g., 5°C, 25°C/60%RH, 40°C/75%RH) and pulled at various time points (e.g., 0 week , 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 6 months, and 12 months) for testing. Controls are tested at 0 weeks, 12 weeks, 6 months, and 12 months.

[00248] Tests that are performed at each stability time extraction include SEC, CEX, Instron, MFI, and Silicone quantification. 1 syringe is tested for Instron, with the expelled material being used for SEC, CEX, injection force measurements and microflow imaging (MFI), and silicone quantification.

EXAMPLE 6 Component Analysis of Syringe filled with Anti-a4ß7 Antibody formulation

[00249] This study explored how various syringe manufacturers, elastomeric plunger (stopper) materials, and the amount of PS80 in the formulation affected the mechanical properties of the system and the stability of the formulation.

[00250] An experiment design was created exploring 3 different syringe manufacturers, 2 different types of plunger material (stopper), and 2 different molar ratios of PS80 to protein. The remainder of the formulation was kept constant at 170 mg/mL protein, 125 mM arginine, 50 mM histidine, 25 mM citrate, and a pH of 6.5. The needle size on these prefilled syringes was 27G^’ or 29G^” thin wall. The experiments carried out are detailed in Table 15.

[00251] The experimental design inputs for the active part of the experiment are shown below in Table 13, while the constants are shown in Table 14. The experimental design was created using the inputs shown in Table 13.

[00252] The list of experiments is shown in Table 10. Table 13. DOE variables and levels with active formulations

[00253] A concentrated formulation of the anti-a4ß7 formulation is concentrated with polysorbate 80 and diluted below 170 mg/mL. The composition of the starting formulation is shown below in Table 16. Table 16. Starting Formulation Buffer Details

[00254] To dilute the material to the desired formulation composition, stock solutions of PS80 in 25 mM Citrate, 50 mM histidine, 125 mM arginine, pH 6.48 are prepared. Table 17. Stock solution details

[00255] The dilution regime for the formulation is detailed in Table 18. Table 18. Dilution detail

[00256] Manipulation is carried out based on the dilution regime and the initial formulation must be weighed, while the other stock solutions can be pipetted volumetrically. The formulations are filtered. 0.5 mL of formulation is aliquoted into as many 1 mL syringes as possible. The syringes are closed by crimping machine with a 2-4 mm bubble. For each time point, there is one syringe stored with the needle down and one syringe stored sideways. Extra syringes are stored with the needle down.

[00257] Syringes are tested at 5, 25 and 40°C in week 2 and month one. Analytical tests (appearance, Instron, pH, osmolality, density, viscosity, SEC, CEX, and Brightwell) are performed initially and then again in 2 weeks at 25 and 40°C and in 4 weeks at 25°C.

EXAMPLE 7 Analysis of closures of subcutaneous containers used in 27 G thin-walled needle syringes filled with anti-a4ß7 antibody formulation

[00258] This study explores how various syringe models with a 27G needle in wall thickness and various plunger (stopper) manufacturers and models affect the mechanical properties and stability of the formulation over time.

[00259] This study explores how the stability of the subcutaneous liquid formulation of anti-a4ß7 in a pre-filled syringe and the mechanical properties of the syringe are affected by the syringe manufacturer and the plunger (stopper) model for syringes with a 27GTW needle. The data generated from this study can determine the container/closure components for the anti-a4ß7 subcutaneous liquid formulation.

[00260] The experimental design inputs are shown below in Table 19, while the constants are shown in Table 20. The experimental design was created using the inputs shown in Table 19.

[00261] The list of experiments to be carried out is shown in Table 21. Table 19. DOE variables and levels with active formulation

[00262] A concentrated formulation of the anti-a4ß7 formulation is concentrated with polysorbate 80 and diluted to below 160 mg/mL. The composition of the initial formulation is shown below in Table 22. Table 22. Initial Formulation Buffer Details

[00263] To dilute the material to the desired formulation composition, stock solutions of PS80 in 25 mM Citrate, 50 mM Histidine, 125 mM Arginine, pH 6.3 are prepared. Table 23. Stock solution details

[00264] The dilution regime for the formulation is detailed in Table 24. Table 24. Dilution detail

[00265] Manipulation is carried out based on the dilution regime and the initial formulation must be weighed, while the other stock solutions can be pipetted volumetrically. The formulations are filtered. 0.5 mL of formulation is aliquoted into as many 1 mL syringes as possible. The syringes are closed by crimping machine with a 2-4 mm bubble. For each time point, there is a syringe stored with needle down (horizontal position).

[00266] Syringes are tested at 5°C, 25°C/60% RH, and 40°C/75% RH for 1 month, 3 months, 6 months, 9 months (optional), 12 months, 18 months and 24 months.

[00267] Liquid formulations are analytically tested (concentration, osmolality, pH, Instron, MFI, SEC, and/or CEX) in months 1, 3, 6, 9, 12, 18, 24 (5°C); months 1, 3, 6, 9, 12, 18, (25°C); months 1, 3, 6, 9, 12, (40°C); and months 1, 3, (40°C).

EXAMPLE 8 Analysis of subcutaneous formulation of anti-a4ß7 antibody in prefilled plastic syringes

[00268] This study is initiated to research the use of plastic syringes as the container/closure system for a subcutaneous anti-a4ß7 antibody formulation. The stability of a representative subcutaneous formulation of anti-a4ß7 antibody in prefilled plastic syringes is studied. The data generated from this study helps the applicability criteria of using a plastic syringe for a subcutaneous liquid formulation of anti-a4ß7 antibody.

[00269] Stability test samples are prepared as shown below. Stability tests are conducted under the storage conditions of 40°C/75% RH, 25°C/60% RH, 5°C.

[00270] Two types of plastic syringes and a glass syringe (Control) in Table 25 are tested with a subcutaneous liquid formulation of anti-a4ß7 antibody shown in Table 26. Table 27 shows the details of each set of samples to be tested in the experiment. Table 25. Plastic syringes

[00271] Prepared anti-a4ß7 subcutaneous liquid formulation is used for this investigation. The formulations are filtered. Sampling the filtered solution for quality testing as a “before filling” sample (Appearance, MFI, DLS). 0.5 mL of formulation is aliquoted into 1 mL plastic syringes. The syringes are closed using a vacuum crimping machine. Syringes are stored with the needle down.

[00272] An initial check is performed to measure pH, osmolality, density, viscosity, and protein concentration. Analytical tests (appearance, SEC(Aggregates, monomer, LMW), CEX(acid, main, basic), sliding force, MFI, DLS, and/or weight) are performed after 1 week at 40°C, 2 weeks, 40°C, 1 month, 5, 25 and 40°C, 3 months, 5 and 25°C, 6 months at 5 and 25°C, 9 months at 5 and 25°C, and 12 months at 5 and 25° W.

[00273] Samples are taken at 1 month, 3 months, 6 months, 9 months and 12 months at 5°C and 25°C. Samples are taken at 1 week, 2 weeks and 1 month at 40°C.

Example 9:

[00274] Samples were analyzed for appearance, injection strength, SEC, CEX, and microflow imaging at 5°C and 25°C at various time points that may have included 0, 1, 3, 6, and 12 months . The stability of the formulation as measured by SEC and CEX was similar to what was discussed in Examples 1 and 2. For injection force testing, sliding force was measured (Table 28). A statistical model determined that the only significant factor affecting sliding force was the syringe manufacturer, where A had a greater sliding force than B, which was more than C (Figure 17). Changes in syringe sliding force for 12 months at 5°C and 6 months at 25°C were less than 10 N, but mostly less than 5 N. Table 28

EXAMPLE 10 Analysis of pre-filled syringe components used in 27 G thin-walled needle syringes filled with anti-a4ß7 antibody formulation

[00275] This study explored how various syringe manufacturers with a thin-walled 27G needle and various manufacturers of plunger (stopper) and elastomeric materials affected the mechanical properties of the prefilled syringe system and the stability of the formulation over time.

[00276] Three different syringe manufacturers and 4 different plunger (stopper) models were tested with 27G^” thin-walled needles and a formulation containing 160 mg/mL protein, 125 mM arginine, 50 mM histidine, 25 mM citrate , 0.2% PS80, at a pH of 6.5. All samples created and tested are shown in Table 29. Table 29. Experimental details

[00277] Samples were analyzed for appearance, injection force, SEC, CEX, and micro-flow imaging at 5°C and 25°C and 40°C at various time points that may have included 0, 1, 3 , 6, and 12 months. The stability of the formulation as measured by SEC and CEX was similar to what was discussed in examples 1 and 2. For injection force testing, release and sliding force were measured. Results at the initial time point are shown in Table 30. Table 30

[00278] A statistical model showed that syringe manufacturers A and C were similar and had a lower sliding force than manufacturer B, while plunger (stopper) E had slightly higher sliding force than the other plunger (stoppers) .

[00279] Generally the initial release forces were similar among all samples that were tested.

[00280] During 12 months at 5°C, 25°C, and 40°C, the sliding force did not change significantly. However, release force for syringes with plunger (stopper) F increased within 12 months at 25°C and 40°C.

EXAMPLE 11 Analysis of anti-a4ß7 antibody formulation in prefilled syringes

[00281] This study determines how varying levels of protein concentration, polysorbate 80 concentration, citrate concentration, and pH affect anti-a4ß7 antibody formulations in a prefilled syringe format.

[00282] Part of the experimental design is created in JMP with a factorial fraction of two levels of protein concentration (60 to 160 mg/mL), pH (6.0 to 6.3), polysorbate 80:protein molar ratio ( 0.723 to 1.5), and citrate concentration (0 to 25 mM). These formulations have a constant concentration of histidine (50 mM) and Arginine (125 mM) (Formulations 1-8). Variations of these formulations with 25 mM Histidine are added (Formulations 9-10).

[00283] An additional set of formulations is developed to explore formulations with no histidine present and only citrate acting as the buffer (Formulations 11-16). The input levels for all formulations are explored in Table 31. The constants used for all formulations are shown in Table 32. Table 31. DOE variables and levels

[00284] Each formulation is generated from an initial stock formulation containing anti-a4ß7 antibody and diluted with various excipient stock solutions. To achieve reasonable dilution volumes, the anti-a4ß7 antibody stock solution used is shown in Table 34. Two different TFF operations are performed to obtain TFF 1 and 2 formulations. A portion of TFF 1 is used in a dialysis to obtain the formulation labeled “Dialysis”. Table 34. Initial Formulation Buffer Details

[00285] For dilution of the material to the desired formulation composition, stock solutions of each excipient in water are prepared at the concentrations specified by Table 35. Table 35. Stock solution details

[00286] The dilution regime for the formulations is detailed in Tables 36 and 37. Table 36. Dilution detail

[00287] Manipulation is carried out based on the dilution regime and the initial formulation is weighed, while the other stock solutions are pipetted volumetrically. The formulations are filtered. 0.5 mL of formulation is aliquoted into as many 1 mL syringes as possible. The syringes are closed using a crimping machine. Syringes are stored with the needle down.

[00288] Liquid formulations are tested analytically (appearance, pH, osmolality, density, DLS, SEC, CEX, and/or Brightwell) initially, and in 1 week, 40°C, 2 weeks, 40°C, 1 month 25 and 40°C, 2 months, 5 and 25°C, 3 months, 5 and 25°C, 6 months, 5 and 25°C, 9 months, 5 and 25°C, and 12 months, 5 and 25°C .

[00289] Specific formulation pulls according to Table 38 are further performed. Table 38: Specific formulation runs

EXAMPLE 12

[00290] A formulation containing 160 mg/mL protein, 50 mM histidine, 25 mM citrate, 125 mM arginine at pH 6.5 was tested for stability in a glass syringe or two different COP plastic syringes. At 5°C and 25°C after 12 months, the amount of aggregates and monomer was comparable between plastic and glass syringes.

Example 13: Bioavailability of Vedolizumab administered by subcutaneous and intramuscular injection

[00291] A phase I study of the bioavailability of vedolizumab administered by subcutaneous and intramuscular injection to healthy male subjects has been completed. A total of 42 healthy men were included in the study. The subjects were divided into three groups (subcutaneous, intramuscular, and intravenous administration) of 14 subjects each. Subjects were given 180 mg of vedolizumab in one day. The dose was reconstituted from a lyophilized formulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH 6.3. For intramuscular and subcutaneous subjects, the dose was divided into two injections of 1.5 ml each. Blood was sampled to determine the plasma concentration of vedolizumab and the bioavailability of vedolizumab in each set of subjects was determined.

[00292] No serious adverse events or significant infections, clinically significant abnormalities, positive subjective/objective RAMP checklists, nor clinically significant ECG findings were reported

[00293] PK/PD modeling and simulation was completed to determine the dose and extravascular dose schedules that result in similar exposures as intravenous doses to maintain desired serum concentrations at minimum levels.

[00294] The absorption profile (FIG. 18) showed that the concentrations of intramuscular and subcutaneous doses generally overlap. There are no apparent gross differences in the absorption profiles of these routes of administration. The absolute bioavailability of vedolizumab after SC injection was approximately 75% and after IM injection it was approximately 80%.

Example 14. Modeling Subcutaneous Dose Regimens

[00295] PK/PD modeling and simulation was completed to determine the dose and extra-vascular dose schedules that result in similar exposures as intravenous doses to maintain desired serum concentrations at minimum levels.

[00296] A final combined data set (data IV, SC and IM) showed two linear compartment models parameterized in terms of clearance (CL) and central volume of distribution (V2), peripheral volume of distribution (V3), a rate extravascular route-dependent absorption constant (KA) and the relative bioavailability (compared to intravenous administration) of extravascular doses (F). IIV terms were included in CL, V2 and V3 with body weight as the only covariate influencing CL and V3 through allometric effect.

[00297] Model acceptability and predictability was demonstrated through initialization parameter estimates, visual predictive checks, and graph fitting suitability. Model analysis identified body weight as a predictor for vedolizumab PK with variability in PK attributed to between-subject and within-subject components.

[00298] Once the model was demonstrated to be suitable for simulation, simulations were performed to evaluate the effect of route of administration (IV, IM, or SC), and evaluate the effect of dosing frequency (weekly, every 2 weeks , every 4 weeks, and every 8 weeks) at minimum concentrations at steady state. Based on these values and the relative bioavailability of vedolizumab after IM and SC administration (F=69.5%), doses were selected to obtain similar trough concentrations as IV doses.

[00299] Simulations modeled doses and schedules to combine intravenous induction and maintenance schedules. Targets were both exposure (area under drug serum concentration-time curve (AUC)) and trough drug concentration. Tables 40-43 provide simulation results. Table 40. Induction Regimen Combining IV AUC for Weeks 0-6

Example 15: Multiple Dose Phase 2a Study

[00300] A multiple dose phase 2a study can evaluate the safety, tolerability and steady state PK of vedolizumab after multiple doses of vedolizumab via the subcutaneous route of administration and to evaluate the relative bioavailability of the subcutaneous regimen compared to the intravenous regimen. The development of HAHA and neutralizing HAHA and the PD effect of multiple doses of vedolizumab after subcutaneous administration can be evaluated.

[00301] Patients with ulcerative colitis with a partial Mayo score of 1-12 and patients with Crohn's disease containing a CDAI of more than 150 may be included in the study. Cohorts may receive an induction regimen of vedolizumab (300 mg) administered IV at weeks 0 and 2, followed by a maintenance regimen of either of these.

[00302] Vedolizumab (300 mg) administered IV every 4 weeks in weeks 6-22.

[00303] Vedolizumab (300 mg) administered IV every 8 weeks on weeks 6-22.

[00304] Vedolizumab (108 mg) administered SC every week in weeks 6-22.

[00305] Vedolizumab (108 mg) administered SC every 2 weeks in weeks 6-22.

[00306] Vedolizumab (165 mg) administered SC every 3 weeks in weeks 6-22.

[00307] Samples can be collected prior to dosing on day 1, and then again on day 1 (12 hours), 2, 3, 5, 8, 15, 29, 43, 127, 127 (12 hours), 128, 129, 131, 134, 141, and 155 to evaluate PK and PD.

Example 16: Long-term clinical experience with Vedolizumab for the treatment of IBD

[00308] A phase 2 open-label safety extension study has been completed to evaluate the long-term pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy of vedolizumab. Patients were aged 18 to 75 years, and had previously participated in an early PK/PD/safety study in patients with ulcerative colitis or had symptoms of IBD for at least 2 months confirmed by endoscopy and/or histopathology and/or radiology. at 36 months of screening.

[00309] All patients received an intravenous dosing regimen of either 2 mg/kg or 6 mg/kg vedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125 mM sodium chloride, 0.05% polysorbate 80, pH 6.0 (long-term storage at -70°C and up to 3 months at -20°C)) on days 1, 15 and 43, followed by one dose every 8 weeks for up to a total of 78 weeks . Patients were treatment-naive ulcerative colitis or Crohn's disease patients, or ulcerative colitis patients who had participated in a previous clinical study.

[00310] Efficacy/quality of life (QoL); partial Mayo score (PMS), Crohn's disease activity index (CDAI), and inflammatory bowel disease questionnaire (IBDQ) were used to evaluate study results.

PK results

[00311] The mean pre-infusion vedolizumab concentrations were dose proportional, and remained stable and detectable throughout the study.

PD Results

[00312] Receptors (%ACT-1 + [CD4+CD45RO HIGH] and % MADCAM+ [CD4+CD45RO HIGH] were almost completely inhibited throughout the study period at all dose levels.

Partial Mayo score

[00313] Mean baseline PMS was higher for treating ulcerative colitis patients without treatments (5.4) than for ulcerative colitis patients in extension (2.3). By day 43, mean PMS showed a pronounced reduction for both untreated and extended ulcerative colitis patients. By day 155, mean scores of the two groups were similar. Average PMS continued to reduce until day 267, and then leveled off.

Crohn's Disease Activity Index

[00314] Average CDAI of CD patients reduced from 294.6 at baseline to 237.7 on Day 43, and continued to reduce until Day 155 (156.1).

IBDQ

[00315] Patients with ulcerative colitis in extension had the highest mean IBDQ scores at baseline. By day 43, average IBDQ scores increased in all three disease groups. Mean IBDQ scores continued to increase over time in all 3 disease groups, reaching a maximum at day 155 for patients with Crohn's disease, and at day 491 for patients with untreated colitis and patients with extended ulcerative colitis.

C-reactive protein

[00316] Both patients with ongoing ulcerative colitis and Crohn's disease showed reduced mean CRP levels throughout day 155 and then leveled off. Patients with untreated ulcerative colitis had a lower mean CRP level at baseline than patients with ulcerative colitis at extension (2.28 v. 7.09). Mean CRP levels of patients with untreated ulcerative colitis remained relatively constant at all time points assessed.

Other safety results

[00317] No systemic opportunistic infections (including PML) were reported during the study. One patient tested positive for JC viremia at a single time point, although he was negative for JCV at all other time points. Three of 72 patients (4%) had positive HAHA results (two of these were transiently positive). The study showed no evidence of liver toxicity, lymphocytosis, or lymphopenia, or any other drug-associated changes. Conclusions

[00318] Vedolizumab administered at 2.0 or 6.0 mg/kg once every 8 weeks for up to 78 weeks achieved target receptor saturations, was associated with durable mean reductions in disease activity, and improved IBDQ scores, was generally safe and well tolerated, and has demonstrated acceptable immunogenicity.

Example 17: Induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis

[00319] A unique study comprising two randomized, double-blind, multicenter studies designed to evaluate the induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis. Demographic and baseline disease characteristics were comparable across all treatment groups.

[00320] The induction study, using intravenous administration, compared placebo against vedolizumab, at a dose of 300 mg reconstituted from a lyophilized formulation of 60 mg/mL of antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80 , 10% sucrose, at pH 6.3, with an assessment point at 6 weeks after 2 doses of vedolizumab.

[00321] The maintenance study, using the same formulation and route of administration as the induction study, compared placebo against vedolizumab dosed every four weeks, and placebo against vedolizumab dosed every eight weeks. The assessment point for this study was at 52 weeks, analyzing the population responding to induction.

[00322] Blood samples were collected to measure vedolizumab concentrations during the study. The mean serum concentration of vedolizumab at the end of the induction phase was 20 to 30 µg/mL. Mean steady-state serum concentrations of vedolizimab after 30 min IV infusion of 300 mg dose administration were between 9 to 13 µg/mL for the q8wks regimen (8-week regimen) and between 35 to 40 µg/mL for the q4wks regimen (4 week regimen). At the end of the infusion, median plasma concentrations of vedolizimab were between 98 and 101 µg/mL for the q8ks regimen and around 129 and 137 µg/mL for the q4 wks regimen.

[00323] Response summaries of induction and maintenance studies are provided in tables 44-47. A significantly greater proportion of patients treated with vedolizumab achieved clinical response, remission, and mucosal healing at 6 weeks compared to placebo (Table 44). 39% of the intention-to-treat population in the induction phase had previous anti-TNFα failure. Clinical response and remission rates were higher in patients on vedolizumab than on placebo among both those with prior anti-TNF failure and those without prior anti-TNF exposure. In preliminary analyzes at week 6, the rates of adverse events (AEs), serious AEs, and adverse events leading to study discontinuation were higher in the placebo group than in the vedolizumab group. A significantly greater proportion of vedolizumab patients than placebo patients achieved clinical remission, mucosal healing, and corticosteroid-free remission at 52 weeks and response and durable remission (Table 45). 32% of the maintenance study population had prior anti-TNFα failure. Clinical remission and durable clinical response rates were higher with vedolizumab than with placebo in both patients with TNF failure and without TNF. In the safety population (N=895) for weeks 0-52, rates of adverse events (AEs), serious AEs, and serious infections were similar between vedolizumab and placebo groups. No increase in opportunistic or enteric infection rates was observed in the vedolizumab group. Table 44: Induction study results - primary and secondary key assessment points

Example 18: Induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis

[00324] A unique study comprising two randomized, double-blind, multicenter studies designed to evaluate the induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis. Demographic and baseline disease characteristics were comparable across all treatment groups.

[00325] The induction study, using intravenous administration, compared placebo against vedolizumab, at a dose of 300 mg reconstituted from a lyophilized formulation of 60 mg/mL of antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80 , 10% sucrose, at pH 6.3, with an assessment point at 6 weeks after 2 doses of vedolizumab.

[00326] The maintenance study, using the same formulation and route of administration as the induction study, compared placebo against vedolizumab dosed every four weeks, and placebo against vedolizumab dosed every eight weeks. The assessment point for this study was at 52 weeks, analyzing the population responding to induction.

[00327] Surprisingly, this study showed that the Q4 and Q8 week groups generated very similar results. Summaries of responses from induction and maintenance studies are provided in Tables 48-51. A significantly greater proportion of patients treated with vedolizumab achieved clinical remission and improved response compared to placebo (Table 48). Improved remission and response rates were greater in vedolizumab than placebo patients among both those with prior anti-TNF failure and those without prior anti-TNF exposure. The rates of adverse events (AEs), serious AEs, and serious infections were similar between the vedolizumab and placebo groups. No increase in rates of opportunistic or enteric infections was observed in the vedolizumab group. Table 48: Induction study results - primary and secondary assessment points

Example 19: Induction of response and remission in patients with moderately to severely active ulcerative colitis

[00328] A randomized, double-blind, placebo-controlled multicenter study was completed to evaluate the induction effect of vedolizumab at doses of 300 mg (reconstituted from a 60 mg/mL formulation of antibody in 50 mM histidine, 125 mM arginine , 0.06% polysorbate 80, 10% sucrose, at pH6.3 which was lyophilized), in patients with TNFa antagonist failure at week 6 (after 2 doses--0 and 2 weeks) and at week 10 (after 3 doses). The study consisted of 416 patients, 75% of whom were TNFa antagonist failures, and 25% of whom were TNFa naïve. Demographics and medication for concomitant IBD were balanced across treatment groups. Baseline disease characteristics were further balanced across treatment groups for baseline disease, except for baseline disease activity.

[00329] The primary endpoint designed for the study was week 6 of remission (%) in the anti-TNF-a antagonist failure population. The secondary key endpoints that were evaluated (sequential testing procedure) were: week 6 remission (%) in the general population, week 10 remission (%) in anti-TNF-a antagonist failure and general population (using Hochberg procedure), week 6 and 10 sustained remission (%) in anti-TNF-a antagonist failure and general population (using Hochberg procedure), and week 6 improved response (%) in anti-TNF-a antagonist failure population The.

[00330] The study showed that patients with TNF-a antagonist failure required 3 doses to induce remission. Remission rates in patients with TNF-α antagonist failure increased between week 6 and week 10, but only for the vedolizumab group (not placebo). Remission rates for TNF-α antagonist-naïve patients did not increase substantially between weeks 6 and 10. Of the TNF-α antagonist failure population with a high degree of disease severity, 43% never responded to a TNF-α antagonist, and 45 % response loss.

Example 20: Stability

[00331] Several different anti-a4ß7 antibody formulations were tested for stability over the course of 6 to 24 months at 5°C (Tables 6 and 7). Formulations containing a pH of 6.0-6.2 showed approximately less than 4% degradation of major species after 6 months and at 24 months. Several different anti-a4ß7 antibody formulations were tested for stability by SEC for up to 24 months (Tables 4 and 5). Formulations with 60 mg/mL protein concentration and containing 25 mM citrate had a change in aggregates of 0.1-0.2% after 2 years, while formulations containing 160 mg/mL protein and 25 mM citrate had a aggregate increase of approximately 0.3% for 2 years. There was a 0.6-1.1% increase in aggregates for formulations containing 60, 110, or 160 mg/mL of citrate-free protein. For the tested formulations containing citrate but without histidine, after 12 months and 24 months, there was approximately 0.30.4% aggregate growth.

Example 21: Determination of vedolizumab in the CD4:CD8 ratio

[00332] Healthy subjects ages 18-45 were treated with a single 450 mg dose of vedolizumab reconstituted from a lyophilized formulation of 10% sucrose and diluted in a 0.9% saline infusion system. Cerebrospinal fluid (CSF) was collected by lumbar puncture before (baseline) and 5 weeks after the single 450-mg dose of vedolizumab. Each subject served as his or her own control.

[00333] A 5-week time point was selected based on a previous study that showed that MS patients treated with natalizumab demonstrated effects on CSF CD4+:CD8+ lymphocyte ratio and reduction in number of brain lesions after just one dose (Stuve et al. al Arch Neurol,2006;63:1383-1387; and further because at 5 weeks, a dose of 450 mg of vedolizumab is sufficient to saturate the target and provides serum concentrations that exceed the estimated steady-state trough levels associated with the phase 3 dosing regimen of 300 mg every 4 weeks .

[00334] Approximately 15 mL of CSF was obtained from each subject for immunophenotyping. CSF samples were included for analysis if they met the following criteria: <10 RBCs/µL per sample (to minimize peripheral blood contamination); negative result for CSF culture; adequate number of T lymphocytes in each flow cytometry sample; and no detection of serum antibodies to vedolizumab.

[00335] Week 5 median (34.80 µg/mL) and individual subjects' serum vedolizumab concentrations (range 24.9-47.9 µg/mL) were greater than the trough concentration at steady state (~24 µg/mL) for the phase 3 dose regimen. A high degree (>90%) of a4ß7 receptor saturation was observed at week 5 as measured by MAdCAM-1-Fc, indicating vedolizumab saturation of its target in time assessment point assessment.

[00336] Vedolizumab was not detected in any CSF sample (detection limit = 0.125 µg/mL).

Effect on numbers and ratio of CD4+ and CD8+ T lymphocytes

[00337] Vedolizumab did not significantly reduce the CD4+:CD8+ ratio (Table 56). None of the subjects had a post-dose CD4+:CD8+ ratio <1 (p < 0.0001 (1-sided t-test)). Vedolizumab did not significantly reduce the number of CD4+ or CD8+ T lymphocytes in CSF. Furthermore, there were no significant changes in % CD4+ T lymphocytes and % CD8+ T lymphocytes (Table 57). Furthermore, no significant changes in peripheral blood WBC, CD4+ and CD8+ memory T lymphocytes (Table 58) were observed. Table 56: Treatment effect on CSF CD4+:CD8+ ratio (evaluable population, n=13)

summary

[00338] Vedolizumab did not affect CSF CD4+ and CD8+ cell counts or CD4+:CD8+ ratio in healthy volunteers after a single dose of 450 mg. None of the subjects had a reduction in the post-dose ratio of CD4+:CD8+ CSF to less than 1. Vedolizumab was not detected in CSF. Furthermore, there was no change observed in total WBCs or subsets of CD4+ and CD8+ memory T lymphocytes in peripheral blood. Target saturation (a4ß7) in the blood occurred in all subjects at the time of endpoint assessment. CSF CD4+ and CD8+ lymphocyte levels and proportion were similar to those previously reported in the literature.

[00339] These results are consistent with lack of effect of vedolizumab on both immune survival in physiological CNS and pathological CNS inflammation of monkeys.

[00340] While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes and details can be prepared therein without departing from the scope of the invention encompassed by the appended claims. Table 59: Sequences

Claims (12)

1. Stable liquid pharmaceutical formulation, characterized by the fact that it comprises 150 to 180 mg/ml of an anti-a4ß7 antibody, 5 mM to 50 mM EDTA, 10 mM to 75 mM histidine, 0.01% to 0.5 % (w/v) polysorbate 80, and 50 mM to 150 mM arginine, wherein the anti-a4ß7 antibody is vedolizumab, and wherein the formulation has a pH of 6.1 to 6.9. 2. Stable liquid pharmaceutical formulation according to claim 1, characterized by the fact that the concentration of polysorbate 80 is 0.1% to 0.3% (w/v). 3. Stable liquid pharmaceutical formulation according to claim 1 or 2, characterized by the fact that the EDTA concentration is 20 mM to 30 mM. 4. Stable liquid pharmaceutical formulation according to any one of claims 1 to 3, characterized in that the histidine concentration is 25 mM to 50 mM. 5. Stable liquid pharmaceutical formulation according to any one of claims 1 to 4, characterized in that it comprises 160 mg/ml vedolizumab, 25 mM EDTA, 50 mM histidine, 0.2% (w/v) of polysorbate 80, and 125 mM arginine, and where the formulation has a pH of 6.5. 6. Manufactured article characterized by the fact that it comprises a container, a stable liquid pharmaceutical formulation, as defined in any one of claims 1 to 5, and instructions for its use. 7. Manufactured article according to claim 6, characterized by the fact that said stable liquid pharmaceutical formulation comprises 108 mg of antibody. 8. Manufactured article according to claim 6, characterized by the fact that the article is a prefilled syringe. 9. Manufactured article according to claim 8, characterized by the fact that the prefilled syringe has a single dose. 10. Manufactured article according to claim 9, characterized by the fact that the dose is 108 mg. 11. Stable liquid pharmaceutical formulation according to any one of claims 1 to 5, or manufactured article according to any one of claims 6 to 10, characterized by the fact that it is for use in the treatment of inflammatory bowel disease. 12. Stable liquid pharmaceutical formulation or manufactured article, according to claim 11, characterized in that the inflammatory bowel disease is Crohn's disease or ulcerative colitis.