Formulation for Anti-α4β7 antibody

Abstract

To provide a formulation for an anti-α4β7 antibody.SOLUTION: A pharmaceutical composition for treating inflammatory bowel disease in a human patient comprises a humanized anti-α4β7 antibody which comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) shown by SEQ ID NO:8, CDR2 shown by SEQ ID NO:9, and CDR3 shown by SEQ ID NO:10, and a light chain variable region comprising a CDR1 shown by SEQ ID NO:11, CDR2 shown by SEQ ID NO:12, and CDR3 shown by SEQ ID NO:13, where the antibody is subcutaneously administered to the human patient at a dose of 108 mg every two weeks.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to formulations for anti-α4β7 antibodies.

[0002] Related Applications This application is a continuation of U.S. Provisional Patent Application 61 / 5444,054, filed October 6, 2011. and priority to U.S. Provisional Patent Application 61 / 481,522, filed May 2, 2011. The entire contents of the aforementioned application are incorporated herein by reference.

[0003] Sequence Listing This application has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Created on April 30, 2012. The ASCII copy is named 92596603.txt and is 16,986 bytes long. The size is. [Background technology]

[0004] Advances in biotechnology have enabled the development of a variety of proteins for medicinal applications using recombinant DNA. Proteins are more effective than traditional inorganic and organic drugs. are larger and more complex (i.e., have multiple functional groups in addition to complex three-dimensional structures) than Formulation of such proteins poses special problems. To achieve this, the formulation must preserve the structural integrity of at least the core sequence of amino acids of the protein. While it is necessary to synthesize the protein, multiple functional groups of the protein must be protected from degradation at the same time. Proteins can suffer from lack of stability and monoclonal antibodies and polyclonal antibodies are not suitable for use with Local antibodies in particular can be relatively unstable (see, for example, Non-Patent Document 1). There are numerous formulation options available, and no single approach or system is suitable for all proteins. It has been reported that several factors should be taken into consideration (e.g. , see Wang et al.).

[0005] A number of characteristics can affect protein stability. Indeed, even in the case of purified antibodies, Body structure can be heterogeneous, which further complicates the formulation of such systems. Excipients included in the formulation preferably minimize potential immune responses.

[0006] In the case of antibodies, preservation of structural integrity is even more important. The protein is susceptible to chemical instability (i.e., the formation of bonds or cleavage that give rise to new chemical entities). processes involving modification of proteins) or physical instability (i.e., changes in the higher-order structure of proteins) Chemical instability can involve, for example, deamidation, isomerization, hydrolysis, , manifested in oxidation, fragmentation, glycan β-elimination or disulfide conversion. The four most common proteins are Degradation pathways include protein fragmentation, aggregation, deamidation and oxidation. Consequences of chemical or physical instability of the compound include a reduction in the effective dosage, e.g., irritation or These include reduced safety of the treatment due to immune reactivity and frequent manufacturing due to short shelf life. .

[0007] Several publications generally disclose various methods of treating inflammatory bowel disease, including the treatment of inflammatory bowel disease. The present invention provides a dosing scheme for the administration of agents designed to treat colon disorders. For example, Patent Document 1 discloses a method for detecting leukocytes in cells expressing mucosal vascular addressin and MAdCAM. The present invention discloses the treatment of diseases associated with the recruitment of leukocytes to the gastrointestinal tract as a result of binding of Patent Document 2 discloses a method for treating a disease associated with leukocyte infiltration in mucosal tissue and an α4β7 inte Human or humanized immunoglobulins or antigen binding molecules having binding specificity for erythrocytes. The document further discloses administration of an effective amount of the fragment to humans. For example, 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mg of immunization per kg of body weight. The study included the following doses: 1) globulin or fragments and various dosing intervals (7, 14, 21, 28, and 30 days). However, the above mentioned patents and publications do not disclose specific formulations of anti-α4β7 antibodies or the present specification. Importantly, the specific doses and dosing regimens described and claimed in the literature are not disclosed. The aforementioned patents relate to the methods of treatment described and claimed herein, which are supported by clinical trial data. The patent does not disclose the formulation, dosage, and administration regimen that would provide [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO96 / 24673 [Patent Document 2] US2005 / 0095238 [Non-patent literature]

[0009] [Non-Patent Document 1] Wang, et al., J. Pharm Sci. 96:1-26 (2007) Summary of the Invention [Problem to be solved by the invention]

[0010] The antibody formulations of the present invention inhibit leukocyte binding to cells expressing MAdCAM. These compounds may be useful in treating inflammatory bowel disease in patients. The invention provides a method for discovering suitable dosages and administration schedules for compounds that are stable and convenient over time. and developing a formulation, preferably a subcutaneous formulation, that produces a stable, therapeutically effective blood level of the antibody formulation. There is an urgent need to [Means for solving the problem]

[0011] The present invention is characterized by its instability, which makes it susceptible to deamidation, oxidation, isomerization and / or aggregation. Antioxidants or chelating agents as excipients useful in formulating anti-α4β7 formulations, and The formulation improves stability and reduces aggregate formation. , slowing down the breakdown of antibodies therein.

[0012] Thus, in a first aspect, the present invention provides a method for treating atopic dermatitis comprising administering to a subject an anti-α4β7 antibody, an antioxidant or a chelating agent, and The present invention relates to a stable liquid pharmaceutical formulation comprising a mixture of at least one free amino acid.

[0013] In some embodiments, a stable liquid pharmaceutical formulation has less than about 1.0% aggregation after 12 months at room temperature. A stable liquid pharmaceutical formulation has less than about 0.2% aggregates after 12 months at room temperature. It may have a formation.

[0014] In some embodiments, the antioxidant or chelating agent is citrate. In the present case, the chelating agent is EDTA.

[0015] In some embodiments, the free amino acids of the formulation are histidine, alanine, arginine, glucan, lysine, glutamic acid, or a combination thereof. The formulation may contain free amino acids between about 100 mM and about 175 mM. The molar ratio of the free amino acid to the antibody may be at least The ratio can also be 250:1.

[0016] The formulation may also contain a surfactant. Surfactants include polysorbate 20, It can be polysorbate 80, a poloxamer, or a combination thereof.

[0017] In some embodiments, the molar ratio of antioxidant to surfactant is from about 3:1 to about 156:1. .

[0018] The formulation may have a pH between about 6.3 and about 7.0. The pH of the formulation may be between about 6.5 and about 7.0. The formulation may be between about 6.1 and about 7.0 or between about 6.2 and 6.8. The pH of the solution may be between 0.5 and 1.

[0019] In some embodiments, the stable liquid pharmaceutical formulation has a concentration of at least about 60 mg / mL to about 160 mg / mL. The formulation contains at least about 160 mg / mL of anti-α4β7 antibody. The formulation may contain about 150 to about 180 mg / mL of the antibody, or It may contain approximately 165 mg / mL of antibody.

[0020] In another aspect, the present invention provides an anti-α4 antibody having at least about 60 mg / mL to about 160 mg / mL. A stable liquid pharmaceutical formulation comprising a β7 antibody, a buffering agent, and at least about 10 mM citrate. The buffering agent can be a histidine buffer.

[0021] In another aspect, the present invention provides an anti-α4 antibody having at least about 60 mg / mL to about 180 mg / mL. A stable liquid pharmaceutical formulation comprising a β7 antibody, a buffer and at least about 5 mM citrate. The buffering agent can be a histidine buffer.

[0022] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of atopic dermatitis, comprising administering to a subject a subject a therapeutically effective amount of an anti-α4β7 antibody having a concentration of at least about 160 mg / mL. The present invention relates to a stable liquid pharmaceutical formulation comprising about 10 mM citrate. It may contain Baet 80.

[0023] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of atopic dermatitis comprising administering to a subject a subject a therapeutically effective amount of an anti-α4β7 antibody at about 160 mg / mL and at least about 5 mg / mL of an anti-α4β7 antibody. The present invention relates to a stable liquid pharmaceutical formulation comprising a citrate salt of M. The formulation further comprises polysorbate 80. It can contain.

[0024] In another aspect, the present invention provides a method for the preparation of a medicament ... The present invention relates to a stable liquid pharmaceutical formulation comprising a mixture of Resorbate 80. The composition may be present in a container such as a cartridge, syringe or autoinjector.

[0025] The anti-α4β7 antibody in the stable liquid pharmaceutical formulation of the present invention can be vedolizumab. The formulations of the invention may be for subcutaneous, intravenous, or intramuscular administration.

[0026] In some embodiments, the formulation can minimize the immunogenicity of the anti-α4β7 antibody.

[0027] In another aspect, the present invention provides a method for administering to a patient in need thereof a stable liquid medicine as described herein. The present invention relates to a method of treating inflammatory bowel disease comprising administering a pharmaceutical formulation comprising: Administering may be subcutaneous. Administering may be self-administering.

[0028] In yet another aspect, the present invention provides a container and a stable liquid pharmaceutical formulation as described herein. , relating to an article of manufacture that includes instructions for use.

[0029] In one aspect, the present invention relates to a method of treating a human patient suffering from inflammatory bowel disease. The method comprises administering to a human patient suffering from inflammatory bowel disease a human α4β7 integrin. administering a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for The humanized immunoglobulin or antigen-binding fragment thereof is administered in one of the following dose regimens: For example, 165 mg of humanized immunized leukemia vaccine administered as a subcutaneous injection every 2 days during the induction phase treatment regimen. (b) six doses of an immunoglobulin or antigen-binding fragment thereof; and (b) six weeks thereafter, e.g., 16 mg / kg / day as a subcutaneous injection every 2 weeks or every 4 weeks as needed in the maintenance phase treatment regimen Seventh and subsequent doses of 5 mg of humanized immunoglobulin or antigen-binding fragment thereof: and wherein said dosing regimen results in clinical response and clinical outcome in inflammatory bowel disease. and further comprising: a humanized immunoglobulin or antigen-binding fragment that binds to the α4β7 complex; and the antigen-binding region is a light chain variable region having the amino acid sequence shown below. The three complementarity determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region are Contains sex determining regions (CDR1, CDR2 and CDR3): Light chain CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:11; Heavy chain: CDR1 SEQ ID NO:1 2. CDR2 sequence number: 13, CDR3 sequence number: 14.

[0030] In one aspect, the present invention relates to a method of treating a human patient suffering from inflammatory bowel disease. The method comprises administering to a human patient suffering from inflammatory bowel disease a human α4β7 integrin. administering a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and a human immunoglobulin or antigen-binding fragment thereof. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of human origin. The following dosing regimens include an intravenous induction phase and a subcutaneous maintenance phase: (a) as an intravenous infusion (b) an initial intravenous administration of 300 mg of humanized immunoglobulin or antigen-binding fragment thereof; Approximately two weeks after the first dose, the humanized immunoglobulin or its antigen-binding protein is administered intravenously. (c) a second intravenous follow-up dose of 300 mg of the combined fragment, starting at week 6, as needed. Humanized immune globulin as a subcutaneous injection every 1, 2, 3, or 4 weeks depending on the and administering to the patient a third and subsequent dose of 165 mg of the compound or an antigen-binding fragment thereof according to the following: the dosing regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and Furthermore, the humanized immunoglobulin or antigen-binding fragment thereof has binding specificity for the α4β7 complex. The antigen-binding region has three complementarity determining domains of the light chain variable region having the amino acid sequence shown below: The three complementarity determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region (C Light chain CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3) Heavy chain: CDR1 SEQ ID NO:12, CDR2 SEQ ID NO:13; Heavy chain: CDR1 SEQ ID NO:14, CDR3 SEQ ID NO:15 Sequence number: 13, CDR3 sequence number: 14.

[0031] In another aspect, the present invention relates to a dosing regimen for the treatment of inflammatory bowel disease. and the administration regimen induces an inhibitory effect against human α4β7 integrin in patients suffering from inflammatory bowel disease. The method includes administering a humanized immunoglobulin or antigen-binding fragment thereof having a binding specificity to the A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and an antigen-binding region of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of the present invention. ~ Mean steady-state trough serum concentration of immunoglobulin or its antigen-binding fragment of approximately 13 μg / mL and administering the drug to the patient according to a subcutaneous or intramuscular dosing regimen that maintains the patient's Induce clinical responses and clinical remission in inflammatory bowel disease; and further, The globulin or antigen-binding fragment thereof has binding specificity for the α4β7 complex; antigen-binding The region is composed of three complementarity determining regions (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR11, CDR19, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19 ...9, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR1 The three complementarity determining regions of the heavy chain variable region (CDR1, CDR2 and CDR3) and and CDR3): Light chain CDR1 SEQ ID NO: 9, CDR2 SEQ ID NO: 10, CDR 3 SEQ ID NO: 11; Heavy chain: CDR1 SEQ ID NO: 12, CDR2 SEQ ID NO: 13, CDR R3 sequence number:14.

[0032] In another aspect, the present invention relates to a dosing regimen for the treatment of inflammatory bowel disease. and the administration regimen induces an inhibitory effect against human α4β7 integrin in patients suffering from inflammatory bowel disease. The method includes administering a humanized immunoglobulin or antigen-binding fragment thereof having a binding specificity to the A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and an antigen-binding region of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of the present invention. Average steady-state titer of 5 to about 40 μg / mL of humanized immunoglobulin or antigen-binding fragment thereof The drug is administered to a patient according to a subcutaneous or intramuscular dosing regimen that maintains a serum concentration of Induces clinical response and clinical remission in inflammatory bowel disease in patients; and the modified immunoglobulin or antigen-binding fragment has binding specificity for the α4β7 complex; The binding region is composed of three complementarity determining regions (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR111, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR111, CDR112, CDR113, CDR114 , CDR2 and CDR3) and the three complementarity determining regions of the heavy chain variable region (CDR1, CDR 2 and CDR3): Light chain CDR1 SEQ ID NO: 9, CDR2 SEQ ID NO: 10, C DR3 SEQ ID NO:11; Heavy chain: CDR1 SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3 sequence number:14.

[0033] In another aspect, the present invention relates to a method of treating a patient suffering from inflammatory bowel disease. The method comprises administering to a patient suffering from inflammatory bowel disease a binding agent for human α4β7 integrin. The method comprises administering a humanized immunoglobulin or an antigen-binding fragment thereof having human specificity to a mammal. The modified immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and an antigen-binding region of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of the humanized immunoglobulin or antigen-binding fragment thereof, Plan: (a) Approximately 20-30 μg / mL of humanized immunoglobulin administered by first dose approximately 6 weeks or an antigen-binding fragment thereof. or an antigen-binding fragment thereof; (b) thereafter, optionally, about 9 to about 13 Average concentration of immunoglobulin or its antigen-binding fragment in μg / mL or about 35-40 μg / mL Multiplexing of humanized immunoglobulins or antigen-binding fragments thereof to maintain normal trough serum concentrations and a maintenance phase dose of at least one of the above, wherein the administration regimen is effective in treating inflammatory bowel disease in the patient. and further comprising administering to said patient a humanized immunoglobulin or its antibody. The antigen-binding fragment has binding specificity for the α4β7 complex; the antigen-binding region is The three complementarity determining regions (CDR1, CDR2, and CDR3) of the light chain variable region of the amino acid sequence and the three complementarity determining regions (CDR1, CDR2 and CDR3) of the heavy chain variable region: 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 .

[0034] In some aspects, the formulations, methods of treatment, dosages and / or dosing regimens are provided to a patient receiving anti-α4β7 antibodies. This ensures a minimal possibility of developing antibodies reactive against the

[0035] Patients may receive at least one of the following treatments: immunomodulatory agents, tumor necrosis factor (TNF-α) antagonists, or a combination of both. Lack of adequate response to at least one treatment , or may not have been tolerated.

[0036] The inflammatory bowel disease may be Crohn's disease or ulcerative colitis. The patient may have moderate to severe active ulcerative colitis.

[0037] The dosing regimen results in mucosal healing in patients suffering from moderately to severely active ulcerative colitis. It brings about healing.

[0038] Patients were previously treated with at least one corticosteroid for inflammatory bowel disease. Patients were concurrently receiving at least one corticosteroid for inflammatory bowel disease. Treatment with steroids may be used. The dosing regimen should be adjusted to accommodate the patient's use of corticosteroids. This results in the reduction, elimination, or reduction and elimination of use.

[0039] In some aspects, the humanized immunoglobulin or antigen-binding fragment thereof has a concentration of about 1.0 mg / mL The humanized immunoglobulin is administered in a final dosage form at a concentration of between about 1.0 mg / mL and about 1.4 mg / mL. Or the antigen-binding fragment thereof can be administered in a final dosage form of about 1.2 mg / mL.

[0040] In some embodiments, the humanized immunoglobulin or antigen-binding fragment is administered in an amount of about 70 to about 250 mg. between about 90 and about 200 mg, between about 150 and about 180 mg, or at least 160 mg The antibody is administered in a final dosage form having an amount of anti-α4β7 antibody of 1000 μg.

[0041] In some embodiments, the dosing regimen includes determining whether CD4+ and CD5+ in the cerebrospinal fluid of patients receiving said treatment are positive for CD4+ and CD5+ in the cerebrospinal fluid of patients receiving said treatment. It does not change the CD8 ratio.

[0042] The patient is a human over 65 years of age and does not require adjustment of the dosing regimen.

[0043] In some aspects, the method, dosage or administration regimen of treatment with the anti-α4β7 antibody formulation comprises administering to the subject an anti-α4β7 The immunogenicity of the antibody can be minimized.

[0044] That is, the gist of the present invention is [1] A pharmaceutical composition for treating inflammatory bowel disease in a human patient, comprising: Complementarity determining region 1 (CDR1) shown in SEQ ID NO: 8, CDR shown in SEQ ID NO: 9 2 and a heavy chain variable region comprising CDR3 shown in SEQ ID NO: 10, and a heavy chain variable region comprising CDR4 shown in SEQ ID NO: 11 CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: and a humanized anti-α4β7 antibody comprising a light chain variable region comprising a pharmaceutical composition for subcutaneous administration to a subject at a dose of 108 mg; [2] A pharmaceutical composition for treating inflammatory bowel disease in a human patient, comprising: Complementarity determining region 1 (CDR1) shown in SEQ ID NO: 8, CDR shown in SEQ ID NO: 9 2 and a heavy chain variable region comprising CDR3 shown in SEQ ID NO: 10, and a heavy chain variable region comprising CDR4 shown in SEQ ID NO: 11 CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: and a humanized anti-α4β7 antibody comprising a light chain variable region comprising: The antibody is administered intravenously to a human patient until an optimal response is achieved, and then 108 mg of the antibody is administered intravenously to the patient. A pharmaceutical composition, wherein a subcutaneous dose is administered to a human patient such that a clinical response is maintained; [3] A clinical response is observed after intravenous administration of a first 300 mg dose of the antibody and 200 mg of the first dose. The pharmaceutical composition of claim 2, wherein the effect is achieved by administering a second 300 mg dose of the antibody two weeks later. thing, [4] A clinical response is observed after administration of a third 300 mg dose of the antibody 4 weeks after the second dose. The pharmaceutical composition according to [3], [5] A subcutaneous dose of 108 mg of the antibody is administered at least 14 weeks after the first dose. The pharmaceutical composition according to [4], [6] Any of [2] to [5], wherein a subcutaneous dose of 108 mg of the antibody is administered every two weeks. The pharmaceutical composition according to any one of claims 1 to 5, [7] A pharmaceutical composition for treating inflammatory bowel disease in a human patient, comprising: has CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:10 A heavy chain variable region comprising the CDR3 shown in SEQ ID NO: 11, and a CDR1 shown in SEQ ID NO: and a light chain variable region comprising CDR2 shown in SEQ ID NO:12 and CDR3 shown in SEQ ID NO:13. The antibody comprises a first intravenous dose of 300 mg of the antibody, a second dose of 300 mg of the antibody administered intravenously two weeks after the first dose, and A 108 mg dose of the antibody administered subcutaneously 6 weeks after dose 1 and every 2 weeks thereafter. a pharmaceutical composition administered according to a dosing regimen comprising: [8] The antibody comprises a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO: 2 and a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO: 4. The pharmaceutical composition according to any one of [1] to [7], comprising a light chain variable region comprising amino acids 20 to 131 of composition, [9] The pharmaceutical composition according to any one of [1] to [8], wherein the antibody is an IgG1 isotype. thing,

[10] The antibody comprises a heavy chain comprising amino acids 20 to 470 of SEQ ID NO: 2 and an amino acid sequence comprising amino acids 20 to 470 of SEQ ID NO: 4. The pharmaceutical composition according to any one of [1] to [7], comprising a light chain containing amino acids 20 to 238.

[11] The pharmaceutical composition according to any one of [1] to [7], wherein the antibody is vedolizumab.

[12] The pharmaceutical composition according to any one of [1] to

[11] , wherein the pharmaceutical composition is prepared for self-administration. Any of the pharmaceutical compositions described above,

[13] The pharmaceutical composition according to any one of [1] to

[12] , wherein the pharmaceutical composition is present in a vial, a cartridge, a syringe, or an auto-injector.

[14] Any of [1] to

[13] , wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. The pharmaceutical composition according to any one of claims 1 to 5,

[15] The method according to

[14] , wherein the ulcerative colitis is moderate to severe active ulcerative colitis. Pharmaceutical compositions,

[16] The administration regimen induces mucosal healing in a human patient with ulcerative colitis. The pharmaceutical composition according to

[14] or

[15] ,

[17] A human patient is treated with at least one corticosteroid for inflammatory bowel disease. The pharmaceutical composition according to any one of [1] to

[16] ,

[18] Patients receiving minimal amounts of immunomodulatory agents, corticosteroids, or tumor necrosis factor-α antagonists have lacked an adequate response to at least one treatment, or have lost response to it or was intolerable to the same. Pharmaceutical compositions,

[19] The administration regimen results in reduction, elimination, or reduction and elimination of the use of corticosteroids. The pharmaceutical composition according to any one of [1] to

[16] , Regarding. Effect of the Invention

[0045] The present invention can provide a formulation for an anti-α4β7 antibody. [Brief description of the drawings]

[0046] [Figure 1A] FIG. 1 shows the nucleotide sequence encoding the heavy chain of a humanized anti-α4β7 immunoglobulin (SEQ ID NO:1) and the deduced amino acid sequence of the heavy chain (SEQ ID NO:2). The nucleotide sequence contains a cloning site (lowercase), a Kozak sequence (uppercase, nucleotides 18-23 of SEQ ID NO:1), and a leader sequence at the 5' end of the heavy chain (lowercase, nucleotides 24-86 of SEQ ID NO:1). The open reading frame of the nucleotide sequence is nucleotides 24-1433 of SEQ ID NO:1. [Figure 1B] Same as above. [Diagram 2]FIG. 1 depicts the nucleotide sequence encoding the light chain of the humanized immunoglobulin referred to herein as vedolizumab (SEQ ID NO:3) and the deduced amino acid sequence of the light chain (SEQ ID NO:4). The nucleotide sequence contains a cloning site (lowercase), a Kozak sequence (uppercase, nucleotides 18-23 of SEQ ID NO:3), and a leader sequence at the 5' end of the heavy chain (lowercase, nucleotides 24-80 of SEQ ID NO:3). The open reading frame of the nucleotide sequence is nucleotides 24-737 of SEQ ID NO:3. [Diagram 3] FIG. 1 shows a sequence comparison of the amino acid sequences of (A) the mature humanized light chain of the humanized immunoglobulin referred to herein as vedolizumab (amino acids 20-238 of SEQ ID NO: 4) and (B) the mature humanized light chain of the humanized immunoglobulin referred to herein as LDP-02 (SEQ ID NO: 5) (for LDP-02 see WO 98 / 06248 and Feagan et al., N. Eng. J. Med. 352:2499-2507 (2005)). Feagan et al. describe clinical trials of LDP-02, but in the paper they refer to LDP-02 as MLN02. The sequence comparison shows that the amino acid sequences of the light chains of vedolizumab and LDP-02 differ at positions 114 and 115 of the mature light chain. [Figure 4] 1 shows an amino acid sequence comparison of (A) the human generic kappa light chain constant region (SEQ ID NO: 6) and (B) the mouse generic kappa light chain constant region (SEQ ID NO: 7). Amino acid residues Thr and Val (located 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 human kappa light chain constant region, whereas amino acid residues Ala and Asp (located at positions 114 and 115 of the mature LDP-02 light chain) (SEQ ID NO: 5) are present in the mouse kappa light chain constant region. [Diagram 5]FIG. 1 shows a map of vector pLKTOK38D (also called pTOK38MLN02-TV), which encodes the humanized heavy and light chains of MLN02 and is suitable for production of vedolizumab in CHO cells (see U.S. Patent Application Publication No. 2004 / 0033561A1, which discloses pLKTOK38; pLKTOK38D is a variant of pLKTOK38 in which mapped restriction sites flank the light chain variable region coding sequence). [Figure 6] The slope of SEC aggregate formation (% per day) as a result of changes in protein concentration, pH, and surfactant:protein molar ratio is shown. In the pH range of 6.0-6.5, aggregate formation is similar to formulations with polysorbate 80:protein in the molar ratio range of 0.7-1.5. [Figure 7] 1 is a graph showing that at polysorbate 80:protein molar ratios above 1.5, the rate of aggregate formation increases with increasing pH. [Figure 8] 1 is a graph showing the effect of excipients on aggregate formation. 25 mM citrate, 5 mM citrate, 5 mM EDTA, 25 mM cysteine, or 5 mM cysteine ​​were added to the formulation. All three excipients reduced aggregate formation. [Figure 9] 1 is a graph showing the reduction of aggregate formation by the presence of 25 mM citrate in the formulation, and the correlation between high protein concentration and high rate of aggregate formation. [Figure 10] 1 is a graph showing the results of the decomposition of CEX species at 40° C. The data shows the effect of pH on CEX decomposition. [Figure 11] 1 is a graph showing the effect of temperature on the pH of the formulations: the pH of the histidine-containing formulations decreases with temperature, while the pH of the citric acid formulation is not affected by temperature. [Figure 12] 1 is a graph showing the proportion of CEX major isoforms over a 12 month period. Formulations with pH 6.0-6.2 showed approximately 1-2% less major isoform than formulations with pH 6.3-6.4. [Figure 13]1 is a set of graphs revealing that viscosity is primarily affected by protein concentration and pH. The addition of sucrose, histidine and arginine is shown to have a minor effect on the viscosity of the formulation. [Figure 14A] FIG. 1 shows the amino acid sequences of (A) the mature human GM607'CL antibody kappa light chain variable region and (B) the human 21 / 28'CL heavy chain variable region. [Figure 14B] Same as above. [Figure 15] FIG. 1 shows the components of a protein product in a pre-filled syringe. [Figure 16] FIG. 1 shows the effect of (A) protein concentration and (B) viscosity on injection force for the various syringes investigated. [Figure 17] (A) Initial glide force as a function of protein concentration and needle size (B) Initial glide force for each syringe manufacturer and needle size. [Figure 18] FIG. 1 shows the absorption profile of vedolizumab. The graph shows that intramuscular and subcutaneous administration concentrations generally overlap. There are no obvious visible differences in the absorption profiles of these routes of administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present invention relates to a pharmaceutical preparation comprising an anti-α4β7 antibody. The mixture contained a stimulant (e.g., citrate), an anti-α4β7 antibody, and a free amino acid. The pharmaceutical formulation may be in solid or liquid form.

[0048] definition The term "pharmaceutical formulation" refers to an anti-α4 antibody in a form that allows the biological activity of the antibody to be effective. Additional agents that contain β7 antibodies and are unacceptably toxic to the subject to which the formulation is administered. Refers to a preparation that does not contain any ingredients.

[0049] A "stable" formulation is one in which the antibody therein maintains its physical stability and / or its chemical stability upon storage. In one embodiment, the preparation substantially retains its inherent stability and / or biological activity. The agent should substantially maintain its physical and chemical stability as well as its biological activity during storage. The storage period is generally selected based on the intended expiration date of the drug product. Various analytical methods for measuring protein stability are available in the art, e.g., Peptide ide and Protein Drug Delivery,247-301,Vi ncent Lee Ed.,Marcel Dekker,Inc.,New York k, NY, Pubs. (1991) and Jones, A. Adv. Drug Del. The temperature selected is reviewed in Livery Rev. 10:29-90 (1993). For example, a liquid formulation can be stored at 40°C for a short period of time. At least about 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In another embodiment, the lyophilized formulation can be stored at 40° C. for at least about 2 to 4 weeks. For each of the following periods: about 3 months, at least about 6 months, at least about 9 months, at least about 12 months The liquid and / or lyophilized formulations are stable for about 18 months or for at least about 18 months. 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, Stable for at least about 24 months, at least about 30 months, or at least about 36 months and / or at -20°C and / or -70°C for at least about 1 month, 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 The compound is also stable for at least about 36 months, at least about 42 months, or at least about 48 months. Further, in some embodiments, the liquid formulations can be frozen (e.g., to -80°C) and thawed. The medium may be stable after, for example, 1, 2 or 3 freeze and thaw cycles.

[0050] The stability of liquid formulations may be assessed by assessment of dimer, multimer and / or aggregate formation (e.g., cytotoxicity). Using size exclusion chromatography (SEC), we have investigated the effect of matrix-assisted laser desorption / ionization spectroscopy. Time-of-flight mass spectrometry (MALDI-TOF-MS), analytical ultracentrifugation, light scattering (photon) correlation Spectroscopic analysis, Dynamic Light Scattering (DLS), Static Light Scattering, Multi-Angle Laser Light Scattering (MALL) S), flow-based microscopy imaging, electronic impedance (Coulter) counting, light obscuration or other liquid particle counting methods, by measuring turbidity and / or by visual inspection) Cation exchange chromatography (CEX), isoelectric focusing (IEF), e.g. Charge heterogeneity is evaluated using capillary electrophoresis (cIEF) or capillary zone electrophoresis. amino- or carboxy-terminal sequence analysis; mass spectrometric analysis; fragmentation, SDS-PAG to compare intact and multimeric (e.g., dimers, trimers, etc.) antibodies E or SEC analysis; peptide map (e.g., trypsin or LYS-C) analysis; antibodies Qualitatively, by a variety of different methods, including evaluating the biological activity or antigen-binding function of the and / or quantitatively assess the stability of solid-state formulations. Identification of crystal structure by X-ray powder diffraction (XRPD); Fourier transform infrared spectroscopy (FT IR) to assess the structure of antibodies in the solid state; and differential scanning calorimetry (DSC) to assess the structure of antibodies in the solid state. The SC method can be used to measure thermal transitions (melting, glass transition, etc.) in freeze-dried solids. as well as extrapolating the potential for chemical instability, e.g., via hydrolysis. In order to determine the moisture content, an indirect test such as the Karl Fisher test is used. The instability can also be assessed qualitatively and / or quantitatively in a variety of different ways, including: The properties include aggregation (e.g., non-covalent soluble aggregation), covalent soluble aggregation (e.g., disulfide reorganization / scrambling of peptide bonds), insoluble aggregation), deamidation (e.g., deamidation of Asn midation), oxidation (e.g., oxidation of Met), isomerization (e.g., isomerization of Asp), cleavage Removal / hydrolysis / fragmentation (e.g., hinge region, fragmentation), succinimide formation, N -terminal extension, C-terminal processing, and / or differences in glycosylation. do.

[0051] A "deamidated" monoclonal antibody is one that has one or more asparagines or glutamines thereof. In some embodiments, the amino acid residues are derivatized to aspartic acid or isoaspartic acid.

[0052] An antibody that is "susceptible to deamidation" has one or more amino acids that are known to be prone to deamidation. It contains residues.

[0053] An antibody that is "susceptible to oxidation" is one that contains one or more residues that are known to be susceptible to oxidation. That is why.

[0054] Antibodies that are "susceptible to aggregation" tend to aggregate more rapidly than antibodies that are "susceptible to aggregation," particularly upon freezing, heating, drying, reconstitution, and / or agitation. It is known to aggregate with other antibody molecules.

[0055] An antibody that is "susceptible to fragmentation" is one that is cleaved, for example, at its hinge region into two or more fragments. It is known that this will happen.

[0056] By "reducing deamidation, oxidation, aggregation or fragmentation," Prevents deamidation, aggregation, or fragmentation compared to monoclonal antibodies formulated in buffers containing Increase or decrease the amount (e.g., 80%, 60%, 50%, 40%, 30%, 20%) or 10%.

[0057] "Aggregates," "SEC aggregates," or "soluble aggregates" are those that are formed by covalent, ionic or loose bonds. Beyond 1, which associates together through aqueous interactions to form even larger protein bodies 10 or less antibody proteins and / or fragments.

[0058] "Insoluble aggregates" or "particles" are formed by the formation of molecules through covalent bonds, ionic bonds or hydrophobic interactions. More than 10 antibody proteins and / or fragments.

[0059] As used herein, the "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen and bind to a test substance. Refers to the ability of an antibody to produce a measurable biological response that can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

[0060] The cell surface molecule, "α4β7 integrin" or "α4β7", binds the α4 chain (CD49D It is a heterodimer of β7 (ITGA4) and β7 (ITGB7). Each chain is involved in the regulation of alternative integrins. It forms heterodimers with the α4β1 or α E β7 can be formed. The human α4 and β7 genes (GenBank (National Center for Biotechnology Information) mation, Bethesda, MD), RefSeq accession numbers NM_000885 and NM_ 000889 is expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes. The integrin α4β7 can normally exist in either a resting or active state. The bonds include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MA). dCAM, for example, MAdCAM-1).

[0061] As used herein, a human immunoglobulin having "binding specificity for the α4β7 complex" Rin or an antigen-binding fragment thereof binds to α4β7 but not to α4β1 or αEβ7 stomach.

[0062] As used herein, an "isotonic" formulation has substantially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of about 250 to 350 mOsm. For example, it can be measured using a vapor pressure type or ice freezing type osmometer.

[0063] As used herein, a "buffering agent" refers to a substance that changes the pH by the action of its acid / base conjugate components. A buffer that is resistant to changes in pH is referred to as a buffer. A buffering agent may be present in the liquid or solid formulation of the present invention. In some embodiments, the buffering agent of the present invention adjusts the pH of the formulation to about 5.0 to about 7.5, at a pH of about 6.0 to about 7.0, or at a pH of about 6.3 to about 6.5 In one embodiment, the pH is adjusted to within the range of 5.0 to 7.5, either alone or in combination. Examples of buffering agents that may be regulated include acetate, succinate, gluconate, histidine, Citrate, phosphate, maleate, cacodylate, 2-[N-morpholino]ethane Sulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxymethyl ]methane (bis-tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine, and other organic acid buffers. The buffer is histidine or citrate.

[0064] A "histidine buffer" is a buffer that contains histidine ions. Examples of histidine buffers Examples include solutions of histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate. The histidine buffer or histidine / HCl buffer has a pH of about 5.5 to about 7.0. The pH range is between about 6.1 and about 6.9, or about 6.5.

[0065] A "citrate buffer" is a buffer that contains citrate ions. Examples of citrate buffers include citrate Dissolved sodium citrate, ammonium citrate, calcium citrate, and potassium citrate Citrate buffer solutions include those with a pH of about 3.0 to 6.2, a pH of about 5.5 to 6.5, and a pH of about It has a pH of 6.1 to about 6.5, about pH 6.1, about pH 6.2 or about pH 6.5.

[0066] "Sugars" as used herein include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, General formula (CH2O) including non-reducing sugars n The present invention relates to a compound having the formula Examples of sugars listed in the literature include glucose, sucrose, trehalose, lactose, and fructose. Sugar, maltose, dextran, erythritol, glycerol, arabitol, silyl ethanol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannose Notriose, Stachyose, Maltose, Lactulose, Maltulose, Glucitol , maltitol, lactitol, iso-maltulose, etc. Sugars are cryoprotectants. In one embodiment, the saccharide referred to herein can be a non-reducing disaccharide, e.g. , sucrose.

[0067] As used herein, "surfactant" refers to an agent that reduces the surface tension of a liquid. In the present specification, the surfactant is a non-ionic surfactant. Sorbates (polyoxyethylene sorbitan monolaurate, e.g., Polysorbate 20 and polysorbate 80); TRITON (t-octylphenoxypolyethoxyethylene Tanol, a nonionic surfactant, from Dow Chemical Company, Midland, Michigan. Carbide Subsidiary); Sodium Dodecyl Sulfate (SDS); Sodium Lauryl Sulfate; Sodium octylglycoside; lauryl, myristyl, linoleyl or stearyl Lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, Coca-amidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmit Propyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl Pil-dimethylamine; sodium methyl cocoyl- or disodium methyl oleyl- Tartaric acid; Sorbitan monopalmitate; and MONAQUAT series (New Jersey Mona Industries, Inc., Paterson, VA; Polyethyl glycol (PEG); Polypropylene glycol (PPG) and poloxyethylene and poloxypropylene glycol copolymers (e.g., Pluronics / Poloxamer, PF68, etc.) In another embodiment, the surfactant herein is polysorbate 80.

[0068] The term "chelating agent" refers to an agent that binds to an atom through more than one bond. Examples of chelating agents herein include citrate, ethylenediaminetetraacetic acid, ethylene Glycoltetraacetic acid (EGTA), dimercaprol, diethylenetriaminepentaacetic acid and N In another embodiment, the chelating agent is chlo- Citrate or EDTA.

[0069] The term "antioxidant" refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants include citric acid, Acid salts, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine , phosphonate compounds such as etidronic acid, desferoxamine and malates; Some examples include:

[0070] The term "antibody" is used in the broadest sense herein and includes human antibodies, humanized antibodies and non-human antibodies. including antibodies derived from different species and recombinant antigen-binding forms such as monofunctional and bifunctional antibodies. , full-length monoclonal antibodies, immunoglobulins, polyclonal antibodies, e.g. Multiple antibodies formed from at least two full-length antibodies each directed against a different antigen or epitope. Multispecific antibodies (e.g., bispecific antibodies), as well as dAbs, scFv, Fab, F(a b) '2. Specific coverage of individual antigen-binding fragments, including Fab'.

[0071] The molar amounts and molar ratios of anti-α4β7 antibody to other excipients described herein are This is calculated assuming an approximate molecular weight of about 150,000 daltons. The molecular weight of an antibody depends on its amino acid composition and post-transcriptional modifications, e.g., on the molecular weight of the cell expressing the antibody. The molecular weight of the actual antibody may vary from 150,000 Daltons depending on the cell line. 000 Daltons ±5%.

[0072] The term "human antibody" includes antibodies derived from transgenic mice carrying human immunoglobulin genes. XENOMOUSE genetically engineered mice (France, CA) Abgenix (Montgomery), HUMAB-MOUSE (registered trademark), KIRIN TC MOUSE™ transchromosomal mice, KMMOUSE® (New Jersey Human germline immunoglobulins such as antibodies derived from MEDAREX (Princeton, VA) globulin sequences, human phage display libraries, human myeloma cells or human B cells Antibodies having sequences derived therefrom are included.

[0073] The term "monoclonal antibody" as used herein refers to a substantially homogeneous population of antibodies. i.e., for example, for variants typically present in minor populations. The population is composed of 100% IgG, ... The individual antibodies that bind different antigens are identical and / or bind the same epitope. In contrast to polyclonal antibody preparations, which contain different antibodies directed against a given base (epitope), Therefore, each monoclonal antibody is directed against a single determinant on the antigen. "Immunotypic" refers to the characterization of an antibody as obtained from a population of substantially homogeneous antibodies. The present invention should not be construed as requiring preparation of the antibody by any particular method. The monoclonal antibodies used in accordance with the invention are described in Kohler et al., Nature, 256: The hybridoma may be produced by the hybridoma method first described by M. K. et al., 495 (1975). or by recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may be produced by, for example, the methods described in Clackson et al., Na ture, 352:624-628 (1991) and Marks et al., J. Mol. Biol. Phage antibody ligation was performed using the technique described in J. Immunol., 222:581-597 (1991). It may be isolated from the lysosome.

[0074] The monoclonal antibodies of the present specification are specifically characterized in that a portion of the heavy and / or light chains is specific to a particular species. Corresponding sequences in antibodies derived from or belonging to a particular antibody class or subclass while the remainder of the chain is from another species or another antibody class. or a "kinase" that is identical or homologous to a corresponding sequence in an antibody belonging to a subclass or subclass. The present invention also includes, but is not limited to, "antibody" or "antibody complexes," as well as fragments of such antibodies, so long as they exhibit the desired biological activity. (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest herein are those derived from non-human primates (e.g. The sequence of the variable domain antigen-binding sequences derived from human monkeys, Old World monkeys, and apes, etc., and the sequence of the human constant regions The antibody includes a "primatized" antibody comprising the sequence.

[0075] The "antigen-binding fragment" of the humanized immunoglobulin prepared in the formulation of the present invention is an anti-α4β7 antibody. For example, the antigen binding domain of vedolizumab includes at least the variable region of the heavy and / or light chains of the human antibody. The humanized light chain fragment contains amino acid residues 20 to 131 of the humanized light chain sequence of SEQ ID NO: 4. Examples of original binding fragments include Fab fragments, Fab' fragments of humanized immunoglobulins known in the art. The humanized immunoglobulin fragments of the present invention include the antibody fragments, scFv and F(ab')2 fragments. The original binding fragments can be produced by enzymatic cleavage or recombinant methods. Fab fragments or F(ab')2 fragments are generated using erythromycin or pepsin cleavage, respectively. It is possible to use an antibody gene in which one or more stop codons have been introduced upstream of the natural stop site. Antibodies can also be produced in various truncated forms, such as F(ab')2 fragments. A recombinant construct encoding the heavy chain of is engineered to encode the CH1 domain and hinge region of the heavy chain. In one embodiment, the antigen-binding fragment can include a DNA sequence encoding the α4β7 inte The mucosal addressin MAdCAM (e.g., M It inhibits binding to AdCAM-1) and fibronectin.

[0076] Papain digestion of antibodies produces fragments called "Fab" fragments, each with a single antigen-binding site. The two identical antigen-binding fragments, and the remaining "Fc", whose name reflects their ability to crystallize Pepsin treatment produces a "fragment" that still has two antigen-binding sites and can bind antigen. F(ab')2 fragments are obtained that are capable of cross-linking.

[0077] "Fv" refers to a peptide that consists of one heavy chain variable domain and one light chain variable domain in a non-covalent association. It is an antibody fragment consisting of a dimer.

[0078] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments consist of heavy chain CH fragments containing one or more cysteines from the hinge region of the antibody. It differs from Fab by the addition of a few residues at the carboxy terminus of one domain. '-SH indicates that the cysteine ​​residues in the constant domain of Fab have at least one free thiol group. F(ab')2 antibody fragments were originally derived from the human F(ab') antibody. The antibody fragments were produced as a pair of Fab' fragments with transcysteines. Coupling is also known.

[0079] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L This includes the domain These domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises Additionally, V allows the scFv to form the desired surface for antigen binding. H and V L domain For a review of scFvs, see Pluckthun et al. in The Pharmacology of Monoclonal Antib odies,vol.113,Rosenburg and Moore eds.,S pringer-Verlag, New York, pp.269-315(1994) Please refer to.

[0080] The term "bifunctional antibody" refers to a small antibody fragment that has two antigen-binding sites. are the same polypeptide chain (V H -V L ) in the variable light chain domain (V L ) connected to a variable weight Chain domain (V H ) that is too short to allow pairing between the two domains on the same chain. A linker is used to force the domains to pair with complementary domains on the other chain, forming two antigens. Bifunctional antibodies are described, for example, in EP 404,097; WO 93 / 111 61; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 9 0:6444-6448 (1993).

[0081] A "full-length antibody" is an antibody that contains the light chain constant domain (C L ) and heavy chain constant domain C H1 , C H2 and C H3 The constant domain comprises a naturally occurring constant sequence. Domains (e.g., human native sequence constant domains) or variants of their amino acid sequences In one embodiment, the full length antibody has one or more effector functions.

[0082] An "amino acid sequence variant" antibody, as used herein, is an antibody that has an amino acid sequence that differs from the primary species antibody. Typically, the amino acid sequence variants have at least about 70% similarity to the major species antibody. At least about 80%, at least about 85%, at least about 90%, or at least about 95% The amino acid sequence variants will have a homology of about 100% to the amino acid sequence of the primary antibody. or adjacent thereto, have substitutions, deletions and / or additions at specific positions, but retain antigen-binding activity. Mutations in the constant region of an antibody are more likely to affect the antigen-binding site than mutations in the variable region. In the variable region, the amino acid sequence variants are less likely to affect the position of the antibody. At least about 90% homology, at least about 95% homology, at least about 97% homology, at least may be about 98% homologous, or at least about 99% homologous.

[0083] "Homology" aligns sequences and introduces gaps if necessary to achieve the maximum percentage of homology. It is defined as the percentage of residues in an amino acid sequence variant that are identical after sequence comparison. Methods and computer programs for this are well known in the art.

[0084] A "therapeutic monoclonal antibody" is an antibody used in the treatment of human subjects. The therapeutic monoclonal antibodies disclosed herein include anti-α4β7 antibodies.

[0085] A "glycosylation variant" antibody, as used herein, is an antibody that has one or more carbohydrate moieties attached to the main species antibody. Glycosylation Mutations: These are antibodies that have one or more carbohydrate moieties linked to them that are different from the original glycosylation mutations. Exemplary antibodies herein include those having G1 or G instead of G0 oligosaccharides linked to their Fc region. Antibodies with two oligosaccharides, one or two carbohydrate moieties linked to one or two of their light chains , antibodies that lack carbohydrate attached to one or both heavy chains of the antibody, etc., and combinations of glycosylation variants. Examples include combinations.

[0086] The "effector functions" of an antibody are determined by the Fc region of the antibody (the native sequence Fc region or amino acid sequence The term refers to those biological activities that result from the Fc region of an antibody (sequence variant). Examples include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, and antibody-dependent cell-mediated ADCC, phagocytosis, cell surface receptors (e.g., B cell receptor, BC R) downregulation.

[0087] Depending on the amino acid sequence of the constant domain of the heavy chain, full-length antibodies are assigned to different "classes". There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG and IgM, some of which are further subdivided (isotypes), e.g., IgG 1. They can be divided into IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of globulins are well known.

[0088] The "light chains" of antibodies derived from vertebrate species are classified into groups based on the amino acid sequence of their constant domains. Each class is assigned to one of two distinct types, called kappa (κ) and lambda (λ).

[0089] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" are mediated by Fc receptors (FcRs). Nonspecific cytotoxic cells expressing IL-1 (e.g., natural killer (NK) cells, leukocytes and macrophages) recognize the bound antibodies on the target cells, which then lyse the target cells ADCC refers to a cell-mediated response that causes ADCC. NK cells are the main cells mediating ADCC. whereas monocytes express only FcγRIII, FcγRI, FcγRII and FcγRII. The expression of FcR on hematopoietic cells was reported by Ravetch and Kinet, An In Table 3 on page 464 of Nu. Rev. Immunol 9:457-92 (1991) To assess the ADCC activity of the molecule, the method described in U.S. Pat. 2 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBM C) and natural killer (NK) cells. Alternatively or additionally, ADCC activity can be measured, for example, by the method described in Clynes et al., PNAS (USA) 95:652-656 (1998), may also be evaluated in vivo in animal models such as those disclosed in

[0090] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In one embodiment, the FcR is a native sequence human FcR. cR binds IgG antibodies (gamma receptors) and is a marker for allelic variants and their FcγRI, FcγRII and FcγRI include alternatively spliced ​​forms of the receptor. FcγRII receptors include the subclass II. FcγRII receptors differ primarily in their cytoplasmic domains. FcγRIIA ("activating receptor") and FcγRIIB, which have similar amino acid sequences ("inhibitory receptors"). The activating receptor FcγRIIA is a It contains an immunoreceptor tyrosine-based activation motif (ITIM) in the cytoplasm. FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. (ITIM) (M. Daeron, Annu. Rev. Immunol. 15 (For review, see Ravetch and K. inet,Annu.Rev.Immunol9:457-92(1991);Cape I et al., Immunomethods 4:25-34 (1994); and de Haa s et al., J. Lab. Clin. Med. 126:33-41 (1995) Other FcRs, including those identified in the future, are encompassed herein by the term "FcR." The term also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus. (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:24 9 (1994).

[0091] The term "hypervariable region" as used herein refers to the amino acids of an antibody that are involved in antigen binding. Hypervariable regions are generally referred to as "complementarity determining regions" or "CDRs" (e.g., the light chain Residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable regions and residues 31-35 (H1), 50-65 (H2), and 95-102 in the heavy chain variable region. (H3);Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) The amino acid residues derived from the IL-11A gene and / or the "hypervariable loops" (e.g., residues in the light chain variable region) Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) and the heavy chain variable region Residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in Chothi a and Lesk J. Mol. Biol. 196:901-917 (1987). The "frame region" or "FR" residues are defined as residues other than the hypervariable region residues as herein defined. The hypervariable regions or CDRs of one antibody chain or another chain or is transferred to another protein to confer antigen-binding specificity to the resulting (composite) antibody or binding protein. Grant.

[0092] Humanized forms of non-human (e.g., rodent) antibodies are the most common form of antibody produced by the synthesis of non-human immunoglobulins. For the most part, humanized antibodies are chimeric antibodies that contain a small amount of sequence. immunoglobulin (recipient), where the residues derived from the recipient hypervariable region are With the desired specificity, affinity and capacity, e.g. mouse, rat, rabbit or non-human Residues are replaced by those from a hypervariable region of a non-human species, such as a primate (donor antibody). In some instances, framework (FR) residues of human immunoglobulins are substituted with corresponding non-human Furthermore, humanized antibodies have the same structure as the recipient antibody or donor antibody, but are replaced with non-human amino acid residues. These modifications may be made to further refine antibody performance. Generally, human A modified antibody contains substantially all of at least one, and usually two, variable domains and has hypervariable loops. All or substantially all of the fragments correspond to those of a non-human immunoglobulin, and all of the FRs or substantially all of the immunoglobulins are human. The antibody will typically comprise at least a portion of a human immunoglobulin constant region (Fc). 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).

[0093] "Affinity matured" antibodies are those that have an increased affinity for an antigen compared to a parent antibody that does not possess these changes. In one embodiment, the antibody has one or more alterations in one or more hypervariable regions thereof that improve the First, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. s et al., Bio / Technology, 10:779-783 (1992) VH and VL describes affinity maturation by replacement of CDR and / or framework residues. The deliberate mutagenesis was carried out as 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(1 995); Jackson et al., J. Immunol. 154(7):3310-9(19 95); and Hawkins et al., J. Mol. Biol. 226:889-896(19 92).

[0094] An "isolated" antibody is one that has been identified and separated from a component of its natural environment and / or In certain embodiments, the antibody is: (1) a IgG antibody that is measured by the Lowry assay; (2) More than 95% by weight of the protein when the spin capsule is By using a peptide sequencer, the N-terminal or internal amino acid sequence of at least 15 residues is obtained. or (3) to a degree sufficient to obtain a stain under reducing conditions using Coomassie blue or silver staining. The isolated fragments may be purified to homogeneity by SDS-PAGE under non-reducing conditions. The antibody produced will have at least one component of the antibody's natural environment absent, such that the antibody will not be present within the recombinant cell. However, isolated antibodies usually include antibodies that have been isolated at least once. It will be prepared during the purification process.

[0095] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. This includes those who already have the disease as well as those whose disease or its recurrence is to be prevented. Thus, the patient treated herein may have been diagnosed with a disease or may be in the process of developing a disease. The terms "patient" and "subject" are used herein to refer to a subject who is susceptible to a disease. are used interchangeably.

[0096] The antibodies that are formulated are substantially pure and preferably substantially homogeneous (free of contaminating proteins). A "substantially pure" antibody is one that is at least 100% pure, based on the total weight of the protein. The present invention relates to compositions that contain at least about 90% by weight, or alternatively at least about 95 or 97% by weight, of antibody. A "substantially homogeneous" antibody has a molecular weight of at least about 99% by total protein weight. A composition comprising a protein in which the amount of the protein is a specific antibody, e.g., an anti-α4β7 antibody. means.

[0097] "Clinical remission," as used herein with respect to a subject with ulcerative colitis, refers to complete A Mayo score of 2 or less and no individual subscores greater than 1. "Clinical remission" in Roan disease refers to a CDAI score below 150.

[0098] "Clinical response," as used herein with respect to a subject with ulcerative colitis, is a score of 1 or more. Complete May with a decrease in the rectal bleeding subscore above or an absolute rectal bleeding score of 1 or less o Score ≥ 3 or 30% decrease from baseline (complete Mayo score in outpatients) If the study was not performed, partial Mayo score of 2 or more points or a 2-point increase from baseline 5% or greater). "Clinical response" is used herein with respect to subjects with Crohn's disease. This is defined as a decrease of 70 points or more in the CDAI score from baseline (week 0).

[0099] "Mucosal healing," as used herein with respect to subjects with ulcerative colitis, is defined as a score of 1 or less. Refers to the endoscopic subscore.

[0100] As used herein, "treatment failure" refers to failure to treat ulcerative colitis or Crohn's disease. Refers to the progression of disease, the need for emergency treatment, or surgical intervention in relation to new or unexplored New medications or treatments required to treat the symptoms of ulcerative colitis or Crohn's disease Increase in dose of baseline medications (other than antidiarrheal agents to control chronic diarrhea).

[0101] formulation As described herein, the anti-α4β7 antibody may be formulated with an antioxidant or a chelating agent. It has been found that the antibody is more stable when the antibody is mixed with the antibody as described herein. The α4β7 antibody can be formulated to reduce aggregate formation (e.g., by using a polysaccharide cleavage inhibitor in the formulation). (The amount of resorbate 80 may be reduced.) For example, citrate or EDTA and anti-α4β7 Antibody-containing formulations reduce the rate of antibody aggregate formation during storage. Store the formulation in the absence of oxygen. In one embodiment, the formulation has a 2.5% retention time after 12 months at 25° C. In one embodiment, the formulation has an antibody aggregate formation of less than about 100% after 12 months at 25° C. In one embodiment, the formulation has an antibody aggregate formation of less than 2.0%. In one embodiment, the formulation has less than about 1.6% antibody aggregate formation after one month at 25° C. After 12 months of administration, the antibody has less than about 1.3% aggregate formation. In one embodiment, the formulation comprises: After 12 months at 25° C., the antibody has less than about 1.0% aggregate formation. In another embodiment, The formulation has less than about 0.5% antibody aggregate formation after 12 months at 5° C. In one embodiment, the formulation has less than about 0.3% antibody aggregate formation after 12 months at 5°C.

[0102] In a first aspect, the present invention provides a stable anti-α4β7 antibody formulation. The formulation also contains a buffer, which may be one or more free amino acids, and an antioxidant or chelating agent. A buffer is also included. The formulation also optionally further includes a surfactant. The antibody in the formulation is a full-length antibody. or, for example, Fab, Fv, scFv, Fab' or F(ab')2 fragments. It may be an antigen-binding fragment thereof.

[0103] Aggregate formation can be reduced by removing oxygen from the formulation. The agent may include an antioxidant or a chelating agent. In one embodiment, the formulation may include Exemplary antioxidants and chelating agents that can be used include lipoic acid, uric acid, glutathione, tocopherol, and erythritol. ferol, carotene, lycopene, cysteine, ethylenediaminetetraacetic acid (EDTA), Ethylene glycol tetraacetic acid (EGTA), dimercaprol, diethylenetriamine pentaacetic acid and N,N-bis(carboxymethyl)glycine, phosphonate compounds such as ethyl Some antioxidants include dronic acid, desferoxamine, malate and citrate. The chelating agent and the stearate agent can reduce the rate of aggregate formation during storage of the formulation. For liquid formulations, the chelating agent and / or antioxidant is citrate or EDTA. Exemplary chelating agent concentrations are 0 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about The range is not more than about 15 mM, about 10 mM to about 25 mM, and about 20 mM to about 30 mM. In another embodiment, the concentration of the chelating agent is from about 0 mM to about 30 mM. The antioxidant and / or antioxidant is citrate, and the concentration of the citrate is 0 mM to about 15 mM. , about 0 mM to about 10 mM, or about 0 mM to about 5 mM.

[0104] The formulation may contain one free amino acid of choice, either in the L- or D-form. or any desired mixture of these forms. In one embodiment, the formulation may include Examples of free amino acids that can be used include histidine, alanine, arginine, glycine, and glutamate. Amino acid, serine, lysine, tryptophan, valine, cysteine, and combinations thereof Some amino acids may be dispersed during production, drying, lyophilization and / or storage. For example, through hydrogen bonds, salt bridges, antioxidant properties or hydrophobic interactions, can stabilize proteins by their exclusion from the protein surface. It may act as a tonicity adjusting agent or to reduce the viscosity of the formulation. In another embodiment, free amino acids such as histidine and arginine can be used as cryoprotectants. It can act as a lyophilisate and does not crystallise when lyophilised as a component of a formulation. Free amino acids such as glutamic acid and histidine, either alone or in combination, are found in 5-7 It can act as a buffering agent in aqueous solutions at pH values ​​in the range of 5. In another embodiment, the formulation comprises histidine, arginine, or a combination of histidine and arginine. In yet another embodiment, the concentration of free amino acids for the liquid formulation is about 9 mM to about 0.5 M, for example, about 10 mM to about 90 mM, about 10 mM to about 75 mM, about 10mM to about 40mM, about 25mM to about 50mM, about 15mM to about 300mM, about 20m M ~ approx. 200mM, approx. 25mM ~ approx. 150mM, approx. 50mM ~ approx. 75mM, approx. 50mM ~ about 120 mM, about 50 to about 150 mM, or within the range of about 50 mM or about 125 mM .

[0105] The formulation may optionally further comprise, for example, at least one additive to control soluble and insoluble aggregate formation. In one embodiment, the surfactant is a non-ionic surfactant. In another embodiment, the surfactant is an ionic surfactant. Exemplary surfactants that can be included include, for example, polysorbate 20, polysorbate Examples of such agents include rubeto 80, poloxamer (Pluronic®), and combinations thereof. When present, surfactants are generally used in silicone, filled vials, pre-filled In the presence of the syringes and / or cartridges, e.g., bottling, freezing, drying, freezing It is included in an amount that reduces the formation of insoluble aggregates of the antibody during lyophilization and / or reconstitution. The concentration of the surfactant is generally about 0.0001% to about 1.0%, 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). High concentrations of surfactants, such as polysorbate 80 Lowering the concentration of polysorbate 80 may result in more SEC aggregate formation. and can reduce SEC aggregate formation during storage. In one embodiment, the surfactant and The molar ratio of the surfactant to the antibody is about 0.7:1 to about 2.0:1. The molar ratio is 1.5:1.

[0106] An embodiment of the anti-α4β7 antibody formulation contains a high concentration of anti-α4β7 antibody. In embodiments, the liquid formulation has a concentration of 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 100 mg / ml ml, at least about 110 mg / ml, at least about 120 mg / ml, at least about 1 30 mg / ml, at least about 140 mg / ml, at least about 150 mg / ml, at least about 160 mg / ml, at least about 170 mg / ml, at least about 180 mg / ml ml, at least about 190 mg / ml, at least about 200 mg / ml, at least about 2 50 mg / ml, at least about 300 mg / ml, from about 60 mg / ml to about 190 mg / ml l, about 60 mg / ml to about 170 mg / ml of anti-α4β7 antibody, about 150 mg / ml to about 180 mg / ml, or about 160 mg / ml or about 165 mg / ml of anti-α4β7 antibody Alternatively, in another embodiment, the liquid formulation may comprise at least about 154 mg / ml, It can contain at least about 176 mg / ml.

[0107] The formulations can be liquid or solid. Liquid formulations are typically prepared by dissolving in water or a water-alcohol mixture. an aqueous solution or suspension prepared in a suitable aqueous solvent, such as an aqueous / organic mixture, The liquid formulation may be, for example, about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 Between about 5.5 and about 7.5, such as 6.6, 6.7, 6.8, or 6.9, between about 6.0 and 7 Between .3, between about 6.0 and about 7.0, between about 6.0 and 6.5, between about 6.0 and 6.3, having a pH between 6.3 and 7.1, or between about 6.4 and 7.0, or between 6.3 and 6.8 Liquid formulations may be stored at room temperature, refrigerated (e.g., 2-8°C), or frozen (e.g., -20°C or - It can be stored at 70°C.

[0108] Solid formulations can be prepared by any suitable method, for example by the addition of cryoprotectants. In one embodiment, the liquid preparations described herein can be in the form of a cream or powder. A solid formulation can be obtained by drying the agent, for example by freeze-drying or spray-drying. If the formulation is a solid formulation, the formulation may contain no more than about 5%, no more than about 4.5%, no more than about or about 4%, only about 3.5%, only about 3%, only about 2.5%, only about 2%, only It can contain about 1.5%, as little as about 1% moisture, or be substantially anhydrous. can be dissolved, i.e., reconstituted in a suitable vehicle to become a liquid suitable for administration. Suitable solvents for reconstituting solid formulations include water, isotonic saline, buffered solutions, For example, phosphate-buffered saline, Ringer's (lactated or dextrose) solution, essential Examples of solvents include mineral media, alcohol / water solutions, and dextrose solutions. The amount of solvent is determined by the dry weight. This may result in a concentration of therapeutic protein that is higher, the same, or lower than the amount prior to drying. The concentration of the reconstituted anti-α4β7 antibody is the same as that of the liquid formulation before drying.

[0109] The formulation may be sterile, which may be suitable for administration to a human subject either before or after preparation of the formulation. This can be accomplished according to procedures known to those skilled in the art for producing sterile pharmaceutical formulations. The formulation may be aseptically processed, for example by filtration through small pores, before drying and / or after reconstitution. It can be sterilized as a liquid through a filter or by exposure to ultraviolet radiation. The pore size of the filter is 0.1 μm to 0.2 μm for filtering microorganisms, and 0.5 μm to filter virus particles. Alternatively or additionally, the dry formulation may have a particle size of 10-20 nm. For example, it can be sterilized by exposure to gamma irradiation. The 4β7 antibody liquid formulation is sterilized by filtration prior to drying.

[0110] In one embodiment, the formulation is stable upon storage. Stability assays for the liquid formulation are available to the skilled practitioner. At least about 4 weeks, at least about 2 months, at least about 3 months, or at least At least about 6 months, or at least about 9 months, or at least about 12 months; 8 o C: at least about 3 months, at least about 1 year, at least about 2 years, at least Alternatively or additionally, the antibody in the formulation may be stable at about 15° C. Storage for at least about 4 weeks, at least about 3 months, at least about 6 months, at least Alternatively or additionally, the formulation may be stable for at least about 9 months, and at least about 1 year or more. The antibodies can be stored at about -20°C or -70°C for at least about 4 weeks; month, at least about 6 months, at least about 9 months, at least about 1 year, at least It may be stable for about 2 years, at least about 3 years, at least about 4 years or more.

[0111] Stability is measured by measuring the stability of the antibody in the formulation before and after formulation as well as after storage at the stated temperatures. by assessing the physical, chemical and / or biological stability. The physical and / or chemical stability of the liquid formulation or reconstituted dry powder may be Assessment of soluble and insoluble aggregate formation (e.g., size exclusion chromatography, analytical ultracentrifugation) Centrifugation, MALDI-TOFMS, light scattering (dynamic (DLS) or MALLS), flow spectrometry by measuring turbidity using microscopic imaging of the sample, or other liquid particle counting methods; by density gradient centrifugation and / or by visual inspection); cation exchange chromatography (Vlas ak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) and Harris et al. J.C. Chromatograms of chromatograms by isoelectric focusing or chromatophoresis (see also Chromatograms of chromatograms by isoelectric focusing or chromatophoresis) By assessing charge heterogeneity using capillary zone electrophoresis; amino-terminus or Carboxy-terminal sequence analysis; mass spectrometric analysis; fragmented, intact and multimeric (e.g. SDS-PAGE analysis to compare antibodies of different structures (e.g. dimers, trimers, etc.); peptide maps A variety of different methods (e.g., Analytical Techniques for Biopharmaceutical Development, Rodriguez-Diaz et al. eds. Inform The assessment can be qualitatively and / or quantitatively carried out in accordance with the International Standards of Health (see International Healthcare (2005)). Instability can be caused by aggregation, deamidation (e.g., deamidation of Asn), oxidation (e.g., M et oxidation), isomerization (e.g., Asp isomerization), denaturation, clipping / hydrolysis / Fragmentation (e.g., fragmentation of the hinge region), formation of succinimides, unpaired cysteines, This can result in N-terminal extension, C-terminal processing, and differences in glycosylation. or antigen-binding function, e.g., MAdCAM (e.g., MAdCA M-1) or MAdCAM (e.g., MAdCAM-1) binding to cells expressing α4β7 integrin For example, MAdCAM-1), for example, immobilized MAdCAM (for example, MAdCAM-1 Inhibition of binding to the agonist can be assessed using a variety of techniques available to the skilled practitioner. (See, e.g., Soler et al., J. Pharmacol. Exper. Ther. 330:864-875 (2009) Measurement of moisture in dry formulations is important to determine how the formulation is subject to chemical or physical degradation. The higher the moisture content, the more rapidly the decomposition proceeds.

[0112] The stable formulation may contribute to the low immunogenicity of anti-α4β7 antibodies. The α4β7 antibody may induce a human / anti-human antibody (HAHA) response in human subjects or patients. Patients who develop a HAHA reaction to anti-α4β7 antibodies may experience adverse events (e.g. For example, the patient may have a site injection reaction, or may be able to rapidly clear the anti-α4β7 antibodies. This can result in lower doses of treatment than planned. Early trials of anti-α4β7 antibody treatment The report by Feagen et al. (2005) N. Engl. J. Med. 352:2499-2507 showed that the showed that 44% of patients developed human anti-human antibodies by 8 weeks. It was stored as a liquid and contained no polysorbates.

[0113] In some embodiments, the formulations exhibit improved HHA results compared to HAHA results for less stable formulations. The proportion of AHA-negative patients should be at least 40%, at least 50%, or at least 60% of patients. %, at least 70%, at least 80%, or at least 90%. do.

[0114] In some embodiments, the anti-α4β7 antibody formulation comprises ≧50% predominant charged isoform, ≥ 55% of the major charged isoforms or 65-70% of the major charged isoforms In other embodiments, the stable anti-α4β7 antibody formulation has ≦45% acidic charged isoforms. ≤40% acidic charged isoforms, ≤30% acidic charged isoforms or 22- In yet another embodiment, the stable anti-α4β7 antibody has 28% acidic charge isoform. The formulations were: ≤25% basic isoform, ≤20% basic isoform, ≤15% about 5% basic isoform or about 10% basic isoform In one embodiment, the stable anti-α4β7 antibody formulation is, for example, prepared by CEX. When measured by , ≥ 55% of the major isoform, ≤ 30% of the acidic isoform, and In another embodiment, e.g., cIEF, As measured by the above, ≥50% of the major isoform, ≤45% of the acidic isoform and and / or ≦10% basic isoform.

[0115] In some aspects, the anti-α4β7 antibody dry solid formulation has ≦10% moisture, ≦5% moisture, or The time required for reconstitution is ≦60 minutes, ≦50 minutes, or ≦4 0 minutes, or ≦30 minutes, or ≦20 minutes.

[0116] Monomer and / or aggregate content (e.g., dimers, etc.) in liquid or reconstituted formulations The chromatographic analysis of chromatographic structures (as trimers, tetramers, pentamers, oligomers and higher aggregates) was carried out using SEC, Ultracentrifugation for analysis, light scattering (DLS or MALLS), MALDI-TOFMS or nanoscale For example, NanoSilicon Tracking Analysis (NTA, Wiltshire, UK) The resolution, characterization, and quantification of aggregates can be performed using a variety of methods, including For example, a second or more SEC columns may be used along with the original analytical SEC column, or a longer column. Increasing the length of SEC column separation by connecting columns in series; In a number of ways, including complementing SEC quantification of monomers, or by using NTA. This can be achieved.

[0117] In one embodiment, the anti-α4β7 antibody formulation is ≧90% monomeric antibody, ≧95% monomeric antibody, In another embodiment, the anti-α4β7 antibody formulation comprises 97% to 99% monomeric antibodies. The majority of the material in the sample has an average diameter of ≦20 nm, ≦15 nm, ≦10 nm, or about 5 to about 7 nm. In one embodiment, the anti-α4β7 antibody preparation has a protein identity of ≧80% by protein analysis. In one embodiment, there are ≧90% heavy and light chains. In another embodiment, there are ≧90% heavy and light chains. , anti-α4β7 antibody preparations had ≦10% aggregates, ≦5% aggregates, ≦2.5% aggregates, ≦1 In another embodiment, the anti-α The 4β7 antibody preparation has ≧96% monomer and / or ≦2.5% aggregates. In embodiments, the anti-α4β7 antibody formulation has ≧99% monomer and / or ≦1% aggregates.

[0118] For example, aggregates or one or more of the undissolved excipients in the liquid formulation or in the reconstituted formulation. Particle sizes above 2 microns are measured using a light obscuration method (e.g., Hach Ultra Ana Liquid particle counting (HIAC) by lytics (Grampus, Oregon), microscope , Coulter counter, or microfluidics by Brightwell (Ottawa, Canada). Methods based on digital (flow-based) microscopic imaging, such as Microscopy Imaging (MFI), or FLOWCAM® Image Particle Analysis by Fluid Imaging Technologies (Yarmouth, Maine). In one embodiment, the amount of α4β7 in an anti-α4β7 antibody preparation can be measured by a chromatograph. The particle size is about 30 μm, about 25 μm, about 10 μm, about 5 μm, about 2 μm, or less than 1 μm. The amount of particles should be reduced as much as possible in the antibody formulation. The amount of particles in the anti-α4β7 antibody formulation is determined based on the single dose package (United States Pharmacopeia, Chp. 788, Photo Obscuration counting method (half the amount by microscopic quantification) <6000 particles ≥10 μm diameter and / or <600 particles ≧25 μm diameter. In another embodiment, one of the anti-α4β7 antibody formulations The amount of particles in the dose was approximately 1000 particles ≧10 μm and 0-100 particles ≧25 μm (MF In yet another embodiment, the method comprises administering to the subject a dose of an anti-α4β7 antibody formulation, for example: The amount of particles per milliliter measured by MFI is approximately 5 per mL for particles between 2 and 10 μm. 00 to about 2000 per mL for particles of ≥ 10 μm, about 50 to about 350 per mL for particles of ≥ 25 μm, In yet another embodiment, the amount of the anti-α4β7 antibody formulation is about 0 to about 50 particles per mL. The amount of particles in the amount is about 500 to about 100,000 particles per mL for 2 to 10 μm particles, and about 1,000 to about 5,000 or about 1,500 to about 3,000.

[0119] The viscosity of the anti-α4β7 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 Increasing the pH can decrease the viscosity of the anti-α4β7 antibody formulation. In some protein formulations, sodium chloride is added to reduce the viscosity of the formulation. Additional components that can affect the viscosity of α4β7 antibody formulations are histidine and arginine. It is an amino acid such as:

[0120] For example, for subcutaneous or intramuscular administration, anti-α4β7 antibody formulations may be isotonic (e.g., 250-350mOsm) or hypertonic (e.g., greater than 350mOsm, 450mOs m, greater than 550 mOsm, greater than 650 mOsm). In one embodiment, the anti-α4β7 antibody formulation is not hypertonic, e.g., is maintained at less than 250 mOsm. In another embodiment, the anti-α4β7 antibody formulation is administered at about 350 to about 400 mOsm, about 400 to about 450 mOsm or about 350 to about 450 mOsm.

[0121] The instability leading to denaturation can be assessed by differential scanning calorimetry (DSC). An antibody has two melting temperatures (Tm) in DSC, e.g., Tm1 and Tm2. Certain excipients may affect the stability of the original anti-α4β7 antibody. The finding of a higher melting temperature when comparing the agents indicates that the more stable anti-α4β7 antibodies have higher Tm. For example, at pH 5.7, the Tm of an anti-α4β7 antibody preparation is low, so It is less stable than pH 6.5. In one embodiment, the Tm1 of the anti-α4β7 antibody formulation is >60 In another embodiment, the Tm1 of the anti-α4β7 antibody formulation is about 65° C. to about 70° C. or about 69° C. In one embodiment, the Tm2 of the anti-α4β7 antibody formulation is >80° C. In other words, the Tm2 of the anti-α4β7 antibody preparation is about 82°C to about 88°C or 86°C.

[0122] In one embodiment, the anti-α4β7 antibody formulation has an affinity for about 60% to about 1% of the anti-α4β7 antibody of the reference standard. In one embodiment, the compounds described herein have a binding affinity or EC50 value of 40%. The anti-α4β7 antibody in the agent is about 80% to about 120% of the reference standard, e.g., in cells (WO98 / 06248 or U.S. Patent No. 7,147,851) binds to α4β7 on In another embodiment, the anti-α4β7 antibody preparation is a cell expressing α4β7 integrin. MAdCAM (e.g., MAdCAM-1), e.g., MAdCAM-Ig chimera (See U.S. Patent Application No. 20070122404, which is also a reference standard.) The inhibitor has the ability to inhibit the activity of the agonist by at least 50% or by at least 60%.

[0123] As mentioned above, freezing of the formulations is specifically contemplated herein. The formulation can be tested for stability upon melting. The antibody may be stable upon freezing and thawing the formulation, e.g., the antibody may be frozen or thawed one, two, three, four, five times. It can be stable after the above freeze-thaw cycles.

[0124] In some embodiments, the pharmaceutical formulation comprises at least about 60 mg / mL to about 170 mg / mL of anti-α4β7 antibody, a buffering agent (e.g., histidine) and at least about 5 mM citric acid. In another embodiment, the formulation is a liquid formulation comprising at least about 60 mg / mL to about 100 mg / mL of the glycerol salt. Approximately 170 mg / mL of anti-α4β7 antibody, a buffer (e.g., citrate), an amino acid ( A liquid preparation containing a surfactant (e.g., polysorbate 80) and It is a drug.

[0125] In another embodiment, the formulation comprises at least about 140 mg / mL or at least about 150 mg / mL About 170 mg / mL, for example, about 160 mg / mL of anti-α4β7 antibody, a buffer (for example histidine), at least 5 mM citrate and free amino acids (e.g., arginine, Contains arginine.

[0126] In yet another embodiment, the formulation comprises at least about 160 mg / mL of anti-α4β7 antibody, A buffer (e.g., histidine), at least 5 mM citrate, 0.2% polyisoprene, In an embodiment, the formulation comprises rubet 80 and a free amino acid (e.g., arginine). The concentration of the buffer solution in the The concentration of the free amino acid in the preparation is about 50 to about 250 mM, about 75 to about 200 mM , about 100 to about 150 mM or about 125 mM; The concentrations are about 0.05%-0.4%, about 0.1%-0.4%, about 0.1%-0.3%, and about 0. 1% to 0.25%, about 0.1% to 0.2%, or about 0.2%.

[0127] In some embodiments, the formulation comprises an anti-α4β7 antibody, citrate, histidine, arginine, Solid formulations containing polysorbate 80 and a cryoprotectant or sugar such as a non-reducing sugar (e.g. Sugar is contained in the liquid formulation at 0% to 20% or about 6% to about 10%. reaches a concentration of

[0128] In one embodiment, the formulation is lyophilized and contained in a single container, e.g., a vial, syringe, capsule, or container. The vial is stored as a single dose in a cartridge, and / or autoinjector. The container can be stored at 2-8° C. or 25° C. until it is administered to a patient in need thereof. The vial can be, for example, a 5, 10, or 20 cc vial (e.g., for a 160 mg / mL dose). The vial can 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-α4β7 antibody. In one embodiment, the container contains about 165 mg of anti-α4β7 antibody.

[0129] In another embodiment, the formulation is a liquid and is dispensed in one or two vials, cartridges, syringes, or dispensers. or in an autoinjector as a single dose. The vial, cartridge, syringe, or autoinjector can be stored at about 2-8° C. until its contents, e.g., anti-α4β7 antibody, are administered to a subject in need thereof. The vial can be, for example, a 5, 10, or 20 cc vial (e.g., for a 160 mg / mL dose). The vial can 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-α4β7 antibody. In one embodiment, the vial contains about 165 mg of anti-α4β7 antibody. The syringe or cartridge can be a 1 mL or 2 mL container (e.g., for a 160 mg / mL dose) or a greater than 2 mL container, e.g., for higher doses (at least 320 mg or 400 mg or more). The syringe or cartridge can 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, or at least about 500 mg of anti-α4β7 antibody.

[0130] Remington:The Science and Practice of P Harmacy, 21st ed., Hendrickson, R. Ed. (2005) One or more other pharma- ceutical acceptable carriers, excipients, or stabilizers such as those mentioned above may be included in the formulation. Acceptable carriers may be included in the formulation provided they do not adversely affect the desired characteristics. The excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed; These include additional buffering agents; co-solvents; antioxidants including citrate and cysteine; EDTA chelating agents such as; metal complexes (e.g., Zn / protein complexes); polyesters such as biodegradable polymers such as silicone; preservatives; container wall lubricants for ease of injection; Mineral oil, glycerin or TRIBOGLIDE® (Tribo Fill) (M Research) perfluoropolyether derivatives and / or sodium Examples of counter ions for salt formation include:

[0131] α4β7 antibody Anti-α4β7 antibodies suitable for use in the formulation include, for example, fully human antibodies, murine antibodies, Antibodies from any desired source, such as rabbit antibodies, etc., and, for example, chimeric antibodies, humanized antibodies The desired engineered antibodies include, for example, Fab, Fv, scFv, Fa, etc. Antigen-binding fragments of any of these types of antibodies, such as F(ab') and F(ab')2 fragments, are also contemplated. Suitable for use in pharmaceutical formulations.

[0132] Anti-α4β7 antibodies target epitopes on the α4 chain (e.g., humanized MAb21.6 (Bendig et al., US Pat. No. 5,840,299), an epitope on the β7 chain (FIB504 or humanized Derivatives (e.g., U.S. Patent No. 7,528,236 to Fong et al.), or α4 and β The present invention is capable of binding to a combined epitope formed by the association of seven chains. In one case, the antibody binds a combination epitope on the α4β7 complex, but the chains do not associate with each other. It does not bind epitopes on the α4 or β7 chains unless Association with integrin brings into close proximity residues present on both chains that together contain the epitope. or on one chain, e.g., the α4 integrin chain or the β7 integrin chain In the absence of an appropriate integrin partner or in the absence of integrin activation, antibody binding by sterically exposing antibody binding sites that are otherwise inaccessible to In another embodiment, the anti-α4β7 antibody can be used to create a peptide that binds to the α4 integrin chain and It binds both β7 integrin chains and is therefore specific for the α4β7 integrin complex. Such an antibody can bind α4β7, but not, for example, α4β1. and / or E In another embodiment, the antibody is capable of binding β7. α4β7 antibody is Act-1 antibody (Lazarovits, AI et al., J. Immunol., 133(4): 1 857-1862 (1984), Schweighoffer et al., J. Immunol., 151(2): 717-729, 1993; rczyk et al., J. Biol. Chem., 269(11): 8348-8354, 1994). Binds to the pitope of mouse Act-1 monoclonal antibody. The -1 hybridoma cell line is registered under the provisions of the Budapest Treaty of August 22, 2001. 40 M Landsdowne St., Cambridge, Massachusetts, USA 02139 2016, for the benefit of Illennium Pharmaceuticals, Inc. American University College, 10801 Boulevard, Manassas, Virginia 110-2209 The strain was deposited in the National Institute of Integrative Medicine at the National Institute of Integrative Medicine under accession number PTA-3663. The anti-α4β7 antibody may be any of the CDRs provided in U.S. Patent Application Publication No. 2010 / 0254975. It is a human antibody or α4β7 binding protein using the method.

[0133] In one embodiment, the anti-α4β7 antibody binds to its ligand (e.g., a mucosal addressin, e.g., For example, MAdCAM (e.g., MAdCAM-1), fibronectin and / or vascular Inhibits the binding of α4β7 to one or more of the vascular endothelial cell adhesion molecule (VCAM). M (e.g., MAdCAM-1) is described in PCT Publication WO96 / 24673. , the entire teachings of which are incorporated herein by reference. The body is able to inhibit the binding of MAdCAM (e.g., MAdCAM-1) without inhibiting the binding of VCAM. ) and / or inhibits the binding of α4β7 to fibronectin.

[0134] In one embodiment, the anti-α4β7 antibody for use in the formulation is a humanized version of the murine Act-1 antibody. Suitable methods for preparing humanized antibodies are well known in the art. The modified anti-α4β7 antibody is a hybridization product of the three heavy chain complementarity determining regions (CDRs, CDRs) of the mouse Act-1 antibody. CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) and a suitable human heavy chain framework region; and CDRs (CDR1, SEQ ID NO: 11, CDR2, SEQ ID NO: 12 and CDR3, SEQ ID NO: 13) and suitable human light chain framework regions. The humanized Act-1 antibody is a humanized antibody that conforms to the consensus framework with or without amino acid substitutions. The polypeptide may contain suitable human framework regions, including framework regions. For example, One or more of the framework amino acids were replaced with the amino acid at the corresponding position in the mouse Act-1 antibody. The human constant region or a portion thereof may be replaced with another amino acid such as If so, the kappa or lambda light chains and / or gamma (e.g., , γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2), δ or ε heavy chains. Certain constant regions (e.g., IgG1), variants or portions thereof can be used to inhibit efflux. For example, a mutated constant region (mutation Incorporating a fusion protein with an Fc receptor binding and / or complement fixing ability (e.g., Winter et al., GB 2,209,757 B; Morrison et al. al., WO 89 / 07142; Morgan et al., WO 94 / 29351, Dec. 22, 1994). Act-1 anti Humanized versions of the construct are described in PCT Publication Nos. WO98 / 06248 and WO07 / 61679. no. 61 / 029,333, the entire teachings of each of which are incorporated herein by reference.

[0135] In another embodiment, the anti-α4β7 humanized antibody for use in the formulation comprises the amino acid sequence of SEQ ID NO:2 A heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO: 4 or amino acids 20 to 131 of SEQ ID NO: 5 and a light chain variable region comprising amino acids 21 to 132 of For example, the humanized anti-α4β7 antibody can have the amino acid sequence 20 of SEQ ID NO:2. The heavy chain may comprise amino acids 21-470 of SEQ ID NO:5 and a light chain may comprise amino acids 21-239 of SEQ ID NO:5. In another example, the humanized anti-α4β7 antibody has 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. A sequence comparison of the common light chains of mouse and human antibodies is shown. Vedolizumab, in which the glycine residues have been replaced by human residues (e.g., Chemical Abstracts Service (CAS, American Chemical Society) registration number 943609-66-3) is the humanized light chain of LDP-02 This explains why the light chain of LDP-02 is more human than that of LDP-03 (Figure 3). In addition, LDP-02 is somewhat hydrophobic. alanine 114, which is hydrophilic and highly flexible, and threonine 114, which is somewhat hydrophilic and contains a hydroxyl group. and vedolizumab, which has a hydrophobic, potentially inward-facing valine residue 115. It has a hydrophilic site (aspartic acid 115) that is

[0136] Further substitutions to the antibody sequence can be made, for example, to the framework regions of the heavy and light chains. For example, a mutation that alters the isoleucine at residue 2 of SEQ ID NO:14 can be used. Mutation of methionine to valine at residue 4 of SEQ ID NO:14; Alanine to glycine mutation at residue 24 of SEQ ID NO:5; alanine to lysine mutation; alanine to arginine mutation at residue 40 of SEQ ID NO:15 Mutation of methionine to isoleucine at residue 48 of SEQ ID NO:15; SEQ ID NO:15 an isoleucine to leucine mutation at residue 69 of SEQ ID NO:15; Mutation of arginine to valine; or combinations thereof; CDRs of the heavy chain CDRs of the mouse Act-1 antibody (CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) and the light chain CDRs of the mouse Act-1 antibody (CDR1, SEQ ID NO: CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13). It is possible.

[0137] In some embodiments, the anti-α4β7 humanized antibody for use in the formulation has the amino acid sequence of SEQ ID NO:2. Approximately 95%, 96%, 97%, 98% or 99% sequence identity for amino acids 2 to 140. A heavy chain variable region having amino acids 20 to 131 of SEQ ID NO: 4 or amino acids 20 to 131 of SEQ ID NO: 5 1 to 132. The amino acid sequence identity is calculated using default parameters, e.g. , Lasergene system (DNASTAR, Madison, Wisconsin) This can be determined using a suitable sequence comparison algorithm. The anti-α4β7 antibody used for this study was vedolizumab (CAS, American Chemical Society, registration number 94360 9-66-3).

[0138] Other α4β7 antibodies may also be used in the formulations and dosing regimens described herein. , US2010 / 0254975 (A The α4β7 antibody described in the Mgen Company is a formulation and and methods for use therein.

[0139] Anti-α4β7 antibodies express the nucleic acid sequences encoding each chain in living cells, e.g., cells in culture. It can be produced by expressing the gene using a variety of host / expression vector systems. Such a host / expression vector system can express an antibody molecule of the invention. The appropriate nucleotide coding sequence represents the medium in which the coding sequence is made and subsequently purified. The present invention represents a cell that can express anti-α4β7 antibody in situ when transformed or transfected with These include, for example, recombinant bacteria containing antibody coding sequences. Transformed with phage DNA, plasmid DNA or cosmid DNA expression vectors recombinant yeast expression vectors containing the antibody coding sequence; Yeast transformed with the vector (e.g., Saccharomyces, Pichia ); recombinant viral expression vectors (e.g. Insect cell lines infected with recombinant vectors (e.g., baculovirus); Recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; or recombinant human ovarian tumor necrosis factor (HV) containing antibody coding sequences. a plant cell line transformed with a plasmid expression vector (e.g., a Ti plasmid); or derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7. A mammalian cell line (e.g., a mammalian cell line harboring a recombinant expression construct containing a 5K promoter) , COS, CHO, BHK, 293, 3T3, NS0) but are not limited to these. For example, the major intermediate-early gene promoter from human cytomegalovirus Chinese hamster ovary cells (CHO) with vectors such as the element Mammalian cells are an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); ockett et al., Bio / Technology 8:2 (1990)).

[0140] In bacterial systems, a number of expression vectors are advantageously selected depending upon the use of the antibody molecule being expressed. For example, large quantities of such proteins can be produced to produce pharmaceutical compositions of antibody molecules. If a fusion protein is to be produced, it is desirable to express high levels of a fusion protein product that is easy to purify. Such vectors may include vectors that direct the expression of fusion proteins. The antibody coding sequence was placed in frame with the lacZ coding region so that The E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983); pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101- 3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)). Examples include, but are not limited to, pGEX vectors that can be used to express glutathione-S-tran. Foreign polypeptides can also be expressed as fusion proteins with GST. Generally, such fusion proteins are soluble and can be expressed in a matrix glutathione / agarose. This is facilitated by adsorption and binding to glutathione beads followed by elution in the presence of free glutathione. The pGEX vector can be used to purify the cloned target gene into a GST region. It is designed to contain thrombin or factor Xa protease cleavage sites to allow release from the will be done.

[0141] In an insect system, Autographa californica has been used as a vector to express foreign genes. ornica nuclear polyhedrosis virus (AcNPV) is used. The antibody coding sequences are individually cloned into the GFP-antibody vector. The AcNPV promoter was cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and The gene is placed under the control of a promoter (eg, the polyhedrin promoter).

[0142] In mammalian host cells, a number of viral-based expression systems are available. When used as an expression vector, the antibody coding sequence is expressed by an adenovirus. The transcription / translation control complex of the present invention can be linked to, for example, the late promoter and tripartite leader sequence. This chimeric gene is then inserted into the adenovirus vector by in vitro or in vivo recombination. It can be inserted into the genome in a non-essential region of the viral genome (e.g., region E1 or E3). The insertion in the vector produces a recombinant virus that is viable in an infected host and capable of expressing the antibody molecule. (e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1999) 84) Specific initiation signals are required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Additionally, the initiation codon must be aligned in phase with the reading frame of the desired coding sequence throughout the entire insert. These exogenous translational control signals and initiation codons must be present to ensure translation of the gene. The expression efficiency depends on the proper transcription factor. The expression of the transcription factor can be enhanced by including enhancer elements, transcription terminators, etc. (Bittner et al. (See, e.g., L., Methods in Enzymol. 153:51-544 (1987)).

[0143] In addition, it is possible to regulate the expression of the inserted sequence and modify the gene product in the specific manner desired, Such modifications of the protein product (e.g., , glycosylation) and processing (e.g., cleavage) are important for protein function. Various host cells may be required for post-translational processing and expression of proteins and gene products. Select an appropriate cell line or host system and Ensure correct modification and processing of the expressed foreign protein. The cells are then transfected with a 50-μl transfection medium to ensure proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Eukaryotic host cells having the machinery for the synthesis of ribozymes can be used. Such mammalian host cells include Chinese Hamster ovary (CHO), NS0, HeLa, VERY, baby hamster kidney (BHK ), monkey kidney (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma cells ( For example, HepG2), for example, BT483, Hs578T, HTB2, BT20 and Breast cancer cell lines such as T47D, and e.g., CRL7030 and Hs578Bst Examples of cell lines that may be used include, but are not limited to, normal breast cell lines such as

[0144] The glycosylation machinery of various cell types allows antibodies to have different glycosylation compositions from different cell types. or, similar to bacteria, antibodies can be produced that are deglycosylated. In one embodiment, the cell type for the production of anti-α4β7 antibodies is NS0 cells or CHO cells. In one embodiment, the mammalian cell is a mammalian cell that is involved in cellular mechanisms. The exogenous gene may include a deletion of an enzyme, for example, by transformation or transfection. Thus, the replacement enzyme is operably linked to a construct or vector for introduction into a cell. The construct or vector with the exogenous gene may be introduced into the construct or vector. A selection preference that induces cells to produce the polypeptide encoded by the construct or vector. In one embodiment, the CHO cells are modified by a deletion or deletion of the dihydrofolate reductase gene. are DG44 cells containing the inactivated gene (Chasin and Urlaub (1980) PNAS USA 77:4216). In another embodiment, the CHO cells are CHO cells containing a deletion or inactivation of the glutamine synthase enzyme. HOK1 cells (see, e.g., U.S. Pat. No. 5,122,464 or U.S. Pat. No. 5,827, (See No. 739).

[0145] Solid Formulations The solid formulations of the present invention are generally prepared by drying a liquid formulation. Drying methods such as freeze drying or spray drying can be used. In freeze-drying, a cryoprotectant is added to the formulation prior to freeze-drying. Containers (e.g., vials, serial numbers, etc.) that will be used to store, ship, and distribute the syringe (e.g., single or dual chamber syringe) or cartridge (e.g., single The method involves drying a liquid formulation in a single or dual chamber cartridge (e.g. 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, The atmospheric pressure is then reduced and the temperature adjusted to remove the frozen solvent, e.g., via sublimation. This step in the freeze-drying process is sometimes called primary drying. The temperature can be raised to remove any solvent still bound to the dried formulation by evaporation. This step in the drying process is sometimes called secondary drying. The drying process is then completed and the container is sealed. The final solid formulation is called a "lyophilized formulation" or "keto-free formulation." The freeze-drying process can be carried out using any suitable equipment. Suitable freeze-drying equipment is available from a number of commercial sources (e.g., NY , SP Scientific, Stone Ridge).

[0146] A variety of suitable devices can be used to dry liquid formulations to form solid (e.g., lyophilized) formulations. Generally, lyophilized formulations can be prepared by immersing a liquid formulation in a vial onto which it is to be dried. The temperature of the shelf is prepared by one skilled in the art using a closed chamber containing a shelf on which the bottle is placed. The temperature can be controlled as can the pressure inside the chamber, as well as the cooling and heating rates. Various process parameters discussed in the specification refer to processes carried out using this type of equipment. It will be understood that those skilled in the art will be able to implement the parameters described herein if desired. It can be easily adapted to other types of drying equipment.

[0147] Suitable temperatures and amounts of vacuum for primary and secondary drying can be readily determined by one of ordinary skill in the art. Generally, the formulations may be frozen at temperatures below about -30°C, e.g., at -40°C or -50°C. The rate of cooling can affect the amount and size of ice crystals in the matrix. Primary drying is generally performed at temperatures about 10°C, about 20°C, about 30°C, about 40°C, or about 50°C below freezing temperature. In one embodiment, the primary drying conditions are at least about 100° C., preferably about 150° C., more preferably about 100° C. Alternatively, the temperature can be set to maintain the anti-α4β7 antibody below the collapse temperature. Above this temperature, the amorphous frozen matrix flows (disintegrates) and the protein molecules become rigid solid matrices. Protein molecules may not be surrounded by trix and may not be stable in the collapse matrix. Also, if collapse does occur, the formulation may not be completely dry. The resulting high water content in the formulation can lead to a high rate of protein degradation and The amount of time that the lyophilized product can be stored before the quality deteriorates to an unacceptable level may be reduced. In one embodiment, the shelf temperature and chamber pressure are maintained below the collapse temperature during primary drying. The glass transition temperature of the frozen formulation is selected to maintain the product at a constant temperature by methods known in the art. Disintegration can be measured by a variety of techniques, for example, by differential scanning calorimetry (DSC). The temperature can be determined by methods known in the art, for example, freeze-drying microscopy, optical coherence The drying process should be at least 50% and at least 60%. In one embodiment, the primary drying step can remove at least 70% or more of the solvent. The process removes greater than 80% of the solvent from the anti-α4β7 antibody composition.

[0148] The size of the vial is selected based on the surface area that will be exposed to the shelf and vacuum during lyophilization. Drying time is directly proportional to cake height, so choose a reasonable cake height. A vial having a large diameter relative to its volume may be selected based on what is determined to be provides high volume contact with the shelves for efficient heat transfer during the freeze-drying cycle. A dilute antibody solution with a large volume will require more time to dry. They are more expensive to store and transport, have a higher headspace to formulation ratio, and have a higher The size of the vial and the volume of the formulation may be affected by the degradative effects of moisture during long-term storage. The 165 mg dose is a good balance between the efficacy and safety of anti-α4β7 antibody preparations. The vial size can be 3 mL, 5 mL, or 10 mL. The size is 5 mL for a 160 mL solution.

[0149] The principles for selecting the size of cartridges or syringes for lyophilization are similar to those for vials As the cake height increases, the drying time will also increase. The diameter and size of the tube must be commensurate with the volume of the final formulation. It increases the rate of water uptake in the freeze-dried cake, enhancing the decomposition effect of water during storage. For the 165 mg dose, the volume of the anti-α4β7 antibody formulation should be 1 mL or 2 mL. In one embodiment, the size of the syringe or cartridge is for a 160 mL solution. The size exceeds 1 mL.

[0150] After lyophilization, the syringe or cartridge is sealed under vacuum, e.g., plugged. Alternatively, a gas, such as dry air or nitrogen, may be introduced into the container prior to sealing. If oxidation is a concern, gas can be admitted to the freeze-drying chamber, A gas that retards or prevents oxidation of the freeze-dried product may be included. In one embodiment, the gas is a non-oxygen-containing gas, such as nitrogen or an inert gas, such as helium, neon, In another embodiment, the gas is nitrogen or argon. do.

[0151] Treatment with antibody preparations In one aspect, the invention relates to a method for the treatment of a disease or disorder, e.g., in a human. The method comprises administering to the subject an anti-α4β7 antibody formulation described herein in an amount sufficient to treat the disease. The present invention provides a method for treating a disease or disorder. The human subject may be an adult (e.g., 18 years of age or older), The human subject may be an adolescent or a child. The human subject may be a human aged 65 years or older. In contrast to the above scheme, human subjects aged 65 years or older may require modifications to the dosing regimen described herein. Instead, a conventional anti-α4β7 antibody formulation as described herein may be administered.

[0152] The subject lacks an adequate response to an immunomodulator, a TNF-α antagonist, or a combination thereof. have had a history of pulmonary embolism, lacked a response to it, or were not tolerant to treatment with it Patients must be receiving at least one corticosteroid for their inflammatory bowel disease. For example, they may have been previously treated with corticosteroids. Inadequate response to steroids was treated with daily oral prednisolone for 2 weeks or intravenous prednisolone for 1 week. Persistent despite experience with at least one 4-week induction regimen including a dose equivalent to 0 mg Lack of response to corticosteroids refers to the signs and symptoms of active disease. Gradually reduce corticosteroids to a dose equivalent to 10 mg of oral prednisolone per day. Corticosteroid intolerance includes Cushing's syndrome, group, osteopenia / osteoporosis, hyperglycemia, insomnia and / or history of infection.

[0153] Immunomodulators include, for example, oral azathioprine, 6-mercaptopurine or methotrexate. Inadequate response to immunomodulators may be due to at least one oral azathioprine. Thiopurine (≥ 1.5 mg / kg), 6-mercaptopurine (≥ 0.75 g / kg) or Despite experience with methotrexate (≥12.5 g / kg) for 8 weeks, Refers to signs and symptoms of active disease. Intolerance to immunomodulators includes vomiting / nausea, abdominal pain, Pancreatitis, LFT abnormalities, lymphopenia, TPMT gene mutations and / or infections. But not limited to these.

[0154] A TNF-α antagonist is, for example, an agent that inhibits the biological activity of TNF-α, preferably TNF -α binding agents, such as monoclonal antibodies, e.g., REMICADE (Invitrogen) fliximab), HUMIRA (adalimumab), CIMZIA (certolizumab pecor) (golimumab), SIMPONI (golimumab), or ENBREL (etanercept) Inappropriate responses to TNF-α antagonists are thought to be due to insulin resistance. Fliximab 5 mg / kg IV in two doses at least 2 weeks apart; adalimumab 80 mg 1 subcutaneous dose of 40 mg, followed by a single dose of 40 mg at least 2 weeks apart; or 400 mg Subcutaneous administration of certolizumab pecor, at least one of two doses at least two weeks apart Refers to signs and symptoms of disease that persist despite undergoing a 4-week induction regimen. Lack of response to NF-α antagonists may be due to recurrence of symptoms during maintenance dosing following previous clinical benefit. Non-tolerance of TNF-α antagonists includes infusion-related reactions, demyelination, congestive heart failure, and and / or infection.

[0155] Loss of maintenance of remission, as used herein for ulcerative colitis patients, is defined as the loss of remission as defined by the Mayo An increase of at least 3 points on the score and at least 2 points on the modified Baron score is considered.

[0156] In another aspect, the present invention provides a method for detecting α4β7 integrin in vitro and / or in vivo. (2) inhibiting the activity or function of α4β7 integrin, e.g., (a) binding to integrin (e.g., α4β7 integrin) and MAdCAM (e.g., MAdCAM-1 ), the ability to bind fibronectin and / or VCAM-1, and / or (b) the ability to bind to tissue Leukocyte infiltration, including recruitment and / or accumulation of leukocytes in the intestinal mucosa (e.g., lymphocyte recruitment to intestinal mucosal tissue) The present invention provides an anti-α4β7 antibody preparation capable of regulating the ability of the antibody to inhibit lymphocyte migration. In one embodiment, the antibody in the formulation is capable of binding α4β7 integrin and signaling (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-1, The present invention can inhibit the binding of α4β7 integrin to one or more of the α4β7 integrin-binding proteins, Thus inhibiting leukocyte infiltration into tissues (including recruitment and / or accumulation of leukocytes in tissues) In another embodiment, the antibody in the formulation is capable of binding α4β7 integrin. , its ligands (e.g., MAdCAM (e.g., MAdCAM-1), VCAM- 1. Selectively inhibiting the binding of α4β7 integrin to one or more of the following: fibronectin This leads to leukocyte infiltration into tissues (recruitment and / or accumulation of leukocytes in tissues). Such anti-α4β7 antibody preparations inhibit the expression of α4β7 in vitro and / or in vivo. Gastrointestinal-associated tissues, lymphoid organs, or white blood cells (especially lymphocytes such as T cells or B cells) Inhibits cell adhesion of cells bearing α4β7 integrin to vascular endothelial cells in mucosal tissues, including In yet another embodiment, the anti-α4β7 antibody formulation of the present invention can be Interaction of MAdCAM (e.g., MAdCAM-1) and / or fibronectin In yet another embodiment, the anti-α4β7 antibody of the present invention can inhibit the activity of The agent may selectively inhibit α4β7 without inhibiting the interaction of α4β7 with VCAM, for example. Interaction of MAdCAM (e.g., MAdCAM-1) and / or fibronectin It can inhibit the use of

[0157] The anti-α4β7 antibody formulation of the present invention is used to inhibit the binding function and / or white blood cells of α4β7 integrin. Regulating (e.g., inhibiting (reducing)) the invasive function of blood cells (e.g., lymphocytes, monocytes) For example, the tissues of white blood cells (e.g., lymphocytes, monocytes), In particular, infiltration of tissues expressing the molecule MAdCAM (e.g., MAdCAM-1) The present invention relates to a method for treating diseases associated with leukocyte recruitment and / or accumulation in tissues. thereby inhibiting the binding of α4β7 integrin to a ligand (i.e., one or more ligands). In one embodiment, a humanized immunoglobulin that is capable of expressing the humanized antibody can be administered.

[0158] To treat such diseases, an effective amount of the anti-α4β7 antibody formulation of the present invention is administered to an individual (e.g., For example, the drug is administered to a mammal, such as a human or other primate, in the gastrointestinal tract (gastrointestinal endothelial cells of the endothelium), other mucosal tissues, or the molecule MAdCAM (e.g., MAdCAM- 1) expressing tissues (e.g., gut-associated tissues such as the venules of the lamina propria of the small and large intestines) In the present invention, inflammatory diseases, including diseases associated with the mammary gland (e.g., lactating mammary gland), are also included. Similarly, MAdCAM (e.g., MAdCAM- 1) is associated with infiltration of leukocytes into tissues as a result of their binding to cells expressing Any individual having a disease can be treated according to the present invention.

[0159] In one embodiment, diseases that can be treated accordingly include, for example, ulcerative colitis, Enteropathy associated with Rohn's disease, ileitis, celiac disease, non-tropical sprue, and seronegative arthropathy ulcerative colitis, microscopic or collagenous colitis, neutrophilic gastroenteritis, or after proctocolectomy Inflammatory bowel disease (IBD) such as pouchitis, and ileoanal anastomosis. In an embodiment, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Ulcerative colitis is Treatment can be moderate to severe active ulcerative colitis. It may result in mucosal healing in patients suffering from colitis. Treatment may also result in corticosteroids being administered by the patient. The reduction, elimination, or reduction and elimination of the use of loids may also occur.

[0160] Pancreatitis and insulin-dependent diabetes mellitus are other conditions that can be treated using the formulations of the present invention. MAdCAM (e.g., MAdCAM-1) is a marker for BALB / c and SJL In the exocrine pancreas derived from NOD (non-obese diabetic) mice as well as from mice, It has been reported that MAdCAM (e.g., MAdCAM-1) is expressed in the Currently, it has been reported that endothelial cells in inflamed islets of the pancreas of NOD mice are induced MAdCAMs (e.g., MAdCAM-1) are expressed in NOD islets at an early stage of insulitis. was the predominant addressin expressed by the epidermis (Hanninen, A., et al., J. Clin. Inv est., 92: 2509-2515 (1993)). Treatment with ruthenium prevented the onset of diabetes (Yang et al., Diabetes, 46:1542-1547 (1997)). Furthermore, accumulation of lymphocytes expressing α4β7 was observed within the islets, and MAdCAM-1 was to the blood vessels of inflamed islets (Hanninen, A., et al., J. Clin. Invest., 92: 2509-251 5 (1993)) or to the gastrointestinal tract in mantle cell lymphoma (Geissmann et al., Am. J. P athol., 153:1701-1705 (1998)) is thought to be involved in the binding of lymphoma cells via α4β7. It was done.

[0161] Examples of inflammatory diseases associated with mucosal tissue that can be treated using the formulations of the present 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, e.g., intestinal venous -Cited disease, or pericholecystitis (tissues surrounding the bile duct and liver), and graft-versus-host disease ( For example, in the gastrointestinal tract (e.g., after bone marrow transplantation) (Petrovic et al. Blood 103:1542-1547 (2004) As seen in Crohn's disease, inflammation can extend beyond the mucosal surface. For example, sarcoidosis, chronic gastritis, autoimmune gastritis (Ka takai et al., Int. Immunol., 14:167-175 (2002)) and other idiopathic conditions. Many inflammatory diseases are amenable to treatment.

[0162] The present invention relates to a method for inhibiting the infiltration of leukocytes into mucosal tissue. The present invention also relates to a method for inhibiting the infiltration of leukocytes into mucosal tissue. The present invention also relates to a method for treating cancers (e.g., α4β7 positive tumors such as lymphomas) by administering the formulations of the present invention to Other examples of inflammatory diseases associated with mucosal tissue that can be treated using the method include mastitis (breast inflammation). ) and irritable bowel syndrome.

[0163] The pathogenesis of this disease relies on the interaction of MAdCAM (e.g., MAdCAM-1) with α4β7. The disease or pathogen to be treated with the anti-α4β7 antibody in the formulation described herein. Examples of such diseases include human immunodeficiency virus (HIV), For example, see WO2008140602).

[0164] The formulations of the invention are administered in an amount effective to inhibit α4β7 from binding to its ligand. For purposes of therapy, an effective amount is an amount sufficient to achieve the desired therapeutic (including prophylactic) effect. would be sufficient (e.g., binding and / or signaling mediated by α4β7 integrin thereby reducing or preventing leukocyte adhesion and infiltration and / or related cellular responses. An effective amount, e.g., an amount sufficient to inhibit α4β7 integrin, e.g., For example, anti-α4β7 antibodies with sufficient titers to maintain neutralization may be clinically effective in inflammatory bowel disease. An effective amount of an anti-α4β7 antibody can induce a favorable response or remission in ulcerative colitis or The formulations of the present invention can be administered in unit or multiple doses to induce mucosal healing in Crohn's disease. The dosage can be determined by methods known in the art. The dosage may depend, for example, on the age, sensitivity, tolerance, and general health of the individual. Exemplary modalities include topical routes, such as intranasal or inhalation or transdermal administration, e.g., intravenous administration, intramuscular administration ... Enteral routes via feeding tubes or suppositories, and also via other routes such as intravenous, intramuscular, subcutaneous, intraarterial, abdominal and parenteral routes, such as intravenous or intravitreal administration. About 0.1 mg / kg to about 10.0 mg / kg of body weight, for example, about 2 mg / kg about 7 mg / kg, about 3 mg / kg to about 6 mg / kg, or about 3.5 mg / kg to about 5 mg / kg In certain embodiments, the dose administered is about 0.3 mg / kg, about 0.5 mg / kg, or about 0.5 mg / kg. / kg, approx. 1mg / kg, approx. 2mg / kg, approx. 3mg / kg, approx. 4mg / kg, approx. 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, or about 1 The total dose is about 22 mg, about 50 mg, about 72 mg, about 125 mg, The total dose may be at least about 77 mg, at least about 165 mg, or at least about 432 mg. In one embodiment, the total dose is at least 165 mg or at least 356 mg. In another embodiment, the total dose is 108 mg. In another embodiment, the total dose is 108 mg. The amount is 216 mg.

[0165] Pharmacokinetic (PK) data from a study of the efficacy of anti-α4β7 antibody over time after administration was used. Modeling and Simulation (University of California, Berkeley Mado NNA™ software is used to evaluate possible dosing regimens for subcutaneous or intramuscular administration. PK data can be evaluated for induction and maintenance regimens. The method of analysis was population pharmacokinetic / pharmacodynamic analysis (ICON PL, Dublin, Ireland). c, NONMEM (registered trademark) nonlinear mixed effects modeling tool). Both plasma and trough levels can be analyzed.

[0166] Typically, after saturation of the target, e.g., α4β7 integrin, is achieved, the antibody concentration in the blood has a linear relationship to the dose administered. Anti-α4 The β7 antibody has a bioavailability of approximately 60% to approximately 90% of that of anti-α4β7 antibody administered intravenously. %. In this relationship example, assume that IV administration has 100% bioavailability, If subcutaneous administration is found to have a bioavailability of 69.5%, then A dose of 300 mg intravenously can be equivalent to a subcutaneous dose of 432 mg. A 216 mg intravenous dose was equivalent to a 216 mg subcutaneous dose with a relative bioavailability of 69.5%. Similarly, subcutaneous administration was found to have a bioavailability of 75%, whereas intramuscular administration is found to have a bioavailability of 80%, then 300 mg The corresponding intravenous dose is 400 mg for subcutaneous administration and 375 mg for intramuscular administration. Tables 40 to 43 in the Examples explain these relationships and show the usefulness of anti-α4β7 antibodies. Dosage and administration schedules are provided.

[0167] In some embodiments, the dosing regimen has two phases, an induction phase and a maintenance phase. For example, inducing immune tolerance to an antibody or antigen-binding fragment thereof or inducing a clinical response. and administering to the patient an effective amount suitable for a particular purpose, such as improving the symptoms of inflammatory bowel disease. The antibody or antigen-binding fragment thereof is provided in a manner that rapidly provides the antibody or antigen-binding fragment thereof. When a patient is being treated with an anti-α4β7 antibody for the first time, the patient is administered anti-α4β7 antibody therapy. For example, more than 3 months, more than 4 months, more than 6 months, more than 9 months since the law was enacted If you have not received treatment for more than one year, more than 18 months, or more than two years, During the maintenance phase of anti-α4β7 antibodies, there is no recurrence of inflammatory bowel disease symptoms, e.g., no disease remission. If there is a relapse from the induction phase, the patient may undergo induction phase treatment. The regimen is designed to achieve a higher mean trough serum concentration than the mean steady-state trough serum concentration maintained during the maintenance regimen. This produces a serum concentration, for example, the concentration immediately prior to the next dose.

[0168] In the maintenance phase, the antibody or antigen-binding fragment thereof is administered to the patient after the induction therapy to obtain a stable antibody or antigen-binding fragment thereof. The maintenance regimen is administered in a manner that maintains a sustained response with levels of antigen-binding fragments of the antibody. Maintenance regimens can prevent recurrence of inflammatory bowel disease or relapse of inflammatory bowel disease. Providing convenience to patients, such as less frequent outpatient visits for treatment or medication In some embodiments, the maintenance regimen may include low dose, small dose, self-administration and pre-administration. by a strategy selected from the group consisting of the above combinations, for example, those described herein The method includes administering an anti-α4β7 antibody or an antigen-binding fragment thereof in a formulation.

[0169] In one embodiment, for example, during the induction phase of the treatment, the dosing regimen is and an effective amount of anti-α4β in the formulations described herein to induce remission of chronic bowel disease. In some embodiments, an effective amount of an anti-α4β7 antibody or antigen-binding fragment is provided. By the end of the induction phase, the blood glucose level should be between about 5 ig / ml and about 60 ig / ml, and between about 15 ig / ml and about 45 ig / ml. l, about 20 ig / ml to about 30 ig / ml, or about 25 ig / ml to about 35 ig / ml of anti-α The duration of the induction phase is approximately 4 The treatment may be for about a week, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks. In this case, the induction regimen may include, for example, the administration of an anti-α4β7 antibody or its derivatives in the formulations described herein. and combinations of high doses, frequent administration, and high doses and frequent administration of the antigen-binding fragment of the present invention. The induction dose may be one or more than one multiple doses. During the induction phase, the dose may be administered once daily, once every two days, or at least twice a day. It may be administered once a day, twice a week, once a week, once every 10 days, once every 2 weeks, or once every 3 weeks. In some embodiments, the induction dose is administered to an anti-α4β7 antibody within the first two weeks of treatment. In one embodiment, the induction dose is administered once at the start of treatment (day 0) and once at the end of treatment. In another embodiment, the duration of the induction phase is six weeks. In another embodiment, the induction phase is six weeks in duration, with multiple induction doses administered during the first two weeks. A dose is administered.

[0170] In some embodiments, for example, patients with severe inflammatory bowel disease (e.g., anti-TNF- When starting treatment with alpha-therapy (patients who have failed alpha-therapy), the induction phase is mild or moderate. In some embodiments, the induction phase of a severe patient should have a longer duration than the induction phase of a severe patient. at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks duration. The induction regimen for patients consisted of a week 0 dose (start of treatment), a week 2 dose, and a week 6 dose. In another embodiment, the induction regimen for a patient with severe disease can include administration of , including week 0 administration (start of treatment), week 2 administration, week 6 administration, and week 10 administration. It is possible to do so.

[0171] In one embodiment, for example, during the maintenance phase of therapy, the dosing regimen is Supernatant concentration, for example, about 5 to about 25 μg / mL, about 7 to about 20 μg / mL, about 5 to about 10 μg / mL g / 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-α4β7 antibody immediately prior to the next dose. Dosage regimens may be, for example, about 20 to about 30 μg / mL, about 20 to about 5 μg / mL, during the maintenance phase of therapy. 5 μg / mL, about 30 to about 45 μg / mL, about 45 to about 55 μg / mL, or about 35 to about 4 0 μg / mL of anti-α4β7 antibody. In this case, the dosing regimen may be, for example, about 15 to about 40 μg / mL during the maintenance phase of therapy, about 10 ~ about 50 μg / mL, about 18 to about 26 μg / mL, or about 22 to about 33 μg / mL of anti-α The mean serum concentration of 4β7 antibody over time, e.g., exposure (e.g., area under the curve-concentration-time In yet another embodiment, the dosing regimen comprises, for example, during the maintenance phase of therapy: About 35 to about 90 μg / mL, about 45 to about 75 μg / mL, about 52 to about 60 μg / mL, or Long-term mean serum concentration of anti-α4β7 antibodies of about 50 to about 65 μg / mL, e.g., after exposure ( For example, area under the curve-concentration-time) is maintained.

[0172] The final dosage form is about 0.5 ml, about 1 ml, about 1.5 ml, or about The total dose may be contained at about 2 ml, at about 2.5 ml, or at about 3 ml.

[0173] The final dosage form for intravenous administration is about 1.0 mg / ml to about 1.4 mg / ml, about 1.0 mg / ml ~ approx. 1.3 mg / ml, approx. 1.0 mg / ml ~ approx. 1.2 mg / ml, approx. 1.0 ~Approx. 1.1mg / ml, approx. 1.1mg / ml ~ approx. 1.4mg / ml, approx. 1.1mg / ml ~1.3mg / ml, approx. 1.1mg / ml ~ approx. 1.2mg / ml, approx. 1.2mg / ml ~ about 1.4 mg / ml, about 1.2 mg / ml to about 1.3 mg / ml, or about 1.3 mg / ml The final dosage form may be at a concentration between about 0.6 mg / ml and about 1.4 mg / ml. , 0.8mg / ml, 1.0mg / ml, 1.1mg / ml, approx. 1.2mg / ml, approx. 1 .3mg / ml, approx. 1.4mg / ml, approx. 1.5mg / ml, approx. 1.6mg / ml, approx. 1 It may be about 0.8 mg / ml or about 2.0 mg / ml.

[0174] Dosages are once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 6 weeks. It can be administered once every 8 weeks, once every 10 weeks, or once every 12 weeks. frequent dosing, e.g., once every 2 days, once per week, once every 2 weeks, once every 3 weeks once every 4 weeks to induce remission of active disease or to treat new patients For example, it may be useful to induce tolerance to anti-α4β7 antibodies. Once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks A dose of once every 10 weeks or once every 10 weeks is appropriate for prophylactic therapy, e.g., in patients with chronic disease. In one embodiment, the treatment regimen includes administering 0 to about 2 In another embodiment, the induction treatment is at about 1 week, about 6 weeks, or every 1-2 weeks thereafter. The plan is one treatment every two days for a total of six treatments.

[0175] The dosing regimen is intended to induce clinical response and clinical remission in patients with inflammatory bowel disease. In some embodiments, the dosing regimen can be optimized for the patient undergoing treatment. Does not change the ratio of CD4 to CD8 in cerebrospinal fluid.

[0176] In some embodiments, a long-lasting clinical remission, e.g., within 6 months or 1 year after initiation of treatment. at least two, at least three, at least four outpatient visits with the attending physician Clinical remission may be achieved with an optimized dosing regimen.

[0177] In some embodiments, a long-lasting clinical response, e.g., at least 6 months after initiation of treatment, Clinical responses lasting at least 9 months and at least 1 year were observed with optimized dosing regimens. This can be achieved.

[0178] The formulation may be administered subcutaneously by a single injection or multiple injections. The volume may range from about 0.5 ml to about 3 ml. In embodiments, the volume of a single injection is about 0.6 ml. The volume of a single injection may be from about 1 ml to about 1.1 ml or from about 1 ml to about 3 ml. In one embodiment, the volume of a single injection is The gauge of the needle used to administer the formulation subcutaneously is about 25, about 26, It may be about 27, about 28, about 29 or about 30G.

[0179] The formulation may be administered intramuscularly by single or multiple injections. The volume may range from about 0.5 ml to about 5 ml. In embodiments, the volume of a single injection is about 2 ml to about 5 ml. It may be about 5 ml, about 0.6 ml to about 1.1 ml, or about 1 ml to about 3 ml. So, the volume of a single injection is about 1 ml, about 2 ml, about 3 ml, about 4 ml or about 5 ml. Needles used to administer the formulation intramuscularly are approximately 5 / 8”, approximately 7 / 8”, approximately 1”, and approximately 1. The gauge of the needle for intramuscular administration can be about 25", about 1.5", about 2" or about 3". It can be between 20 and 22G.

[0180] In one aspect, the present invention relates to a method of treating a human patient suffering from inflammatory bowel disease. The method includes the step of: The method comprises administering the globulin or an antigen-binding fragment thereof to a human patient suffering from inflammatory bowel disease. The humanized immunoglobulin or antigen-binding fragment thereof may be administered in the following dosage regimen: (a) once daily Six doses of 165 mg of humanized immunoglobulin or its antigen-binding fragment administered subcutaneously (b) 165 mg / kg / day as a subcutaneous injection every 2 weeks or 4 weeks thereafter, as needed mg of the seventh and subsequent doses of the humanized immunoglobulin or antigen-binding fragment thereof, and administering to the patient a clinical response or clinical remission in the patient's inflammatory bowel disease. and further comprising: a humanized immunoglobulin or an antigen-binding fragment thereof that is capable of inducing a β4β7 complex-specific α4β7 ... and the antigen-binding region is composed of three light chain variable regions having the amino acid sequences shown below. The three complementarity determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region Contains constant regions (CDR1, CDR2 and CDR3): Light chain CDR1 SEQ ID NO: 9, CD R2 SEQ ID NO:10, CDR3 SEQ ID NO:11; Heavy chain: CDR1 SEQ ID NO:12, CDR2 sequence number: 13, CDR3 sequence number: 14.

[0181] In one aspect, the present invention relates to a method of treating a human patient suffering from inflammatory bowel disease. The method comprises administering to a human patient suffering from inflammatory bowel disease a human α4β7 integrin. administering a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and a human immunoglobulin or antigen-binding fragment thereof. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of human origin. The following dosing regimens: (a) humanized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion (b) an initial dose of 300 mg intravenously, followed by (c) a dose of humanized immunoglobulin A as an intravenous infusion. (c) a second intravenous subsequent administration of 300 mg of phosphorus or an antigen-binding fragment thereof, at week 6 thereafter. Humanized as subcutaneous injections every 1, 2, or 3 weeks as needed, starting at followed by a third subsequent dose of 165 mg of immunoglobulin or an antigen-binding fragment thereof. wherein the dosing regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further comprising: a humanized immunoglobulin or an antigen-binding fragment thereof that binds to the α4β7 complex. The antigen-binding region has the complementarity of the three light chain variable regions of the amino acid sequences shown below. Complementarity determining regions (CDR1, CDR2 and CDR3) of the heavy chain variable region and the three complementarity determining regions (CDR1, CDR2 and CDR3): Light chain CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:11; Heavy chain: CDR1 SEQ ID NO:12, CDR CDR2 SEQ ID NO:13, CDR3 SEQ ID NO:14.

[0182] In another aspect, the present invention relates to a dosing regimen for the treatment of inflammatory bowel disease. and the administration regimen induces an inhibitory effect against human α4β7 integrin in patients suffering from inflammatory bowel disease. The method includes administering a humanized immunoglobulin or antigen-binding fragment thereof having a binding specificity to the A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and an antigen-binding region of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of the present invention. ~ Mean steady-state trough serum concentration of immunoglobulin or its antigen-binding fragment of approximately 13 μg / mL and administering the drug to the patient according to a subcutaneous or intramuscular dosing regimen that maintains the patient's Induce clinical responses and clinical remission in inflammatory bowel disease; and further, The globulin or antigen-binding fragment thereof has binding specificity for the α4β7 complex; antigen-binding The region is composed of three complementarity determining regions (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR11, CDR19, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19 ...9, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR1 The three complementarity determining regions of the heavy chain variable region (CDR1, CDR2 and CDR3) and and CDR3): Light chain CDR1 SEQ ID NO: 9, CDR2 SEQ ID NO: 10, CDR 3 SEQ ID NO: 11; Heavy chain: CDR1 SEQ ID NO: 12, CDR2 SEQ ID NO: 13, CDR R3 sequence number:14.

[0183] In another aspect, the present invention relates to a dosing regimen for the treatment of inflammatory bowel disease. and the administration regimen induces an inhibitory effect against human α4β7 integrin in patients suffering from inflammatory bowel disease. The method includes administering a humanized immunoglobulin or antigen-binding fragment thereof having a binding specificity to the A humanized immunoglobulin or antigen-binding fragment thereof comprises an antigen-binding region of non-human origin and an antigen-binding region of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of an antibody of the present invention. Steady-state serum titers of 5 to about 40 μg / mL of humanized immunoglobulin or its antigen-binding fragments. The drug is administered to a patient according to a subcutaneous or intramuscular dosing regimen that maintains a consistent concentration of the drug in the patient's blood, Induce clinical responses and clinical remission in inflammatory bowel disease in subjects; and further, The immunoglobulin or antigen-binding fragment has binding specificity for the α4β7 complex; antigen binding The region is composed of three complementarity determining regions (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR11, CDR19, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19 ...9, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR1 The three complementarity determining regions of the heavy chain variable region (CDR1, CDR2 and CDR3) and and CDR3): Light chain CDR1 SEQ ID NO: 9, CDR2 SEQ ID NO: 10, CDR 3 SEQ ID NO: 11; Heavy chain: CDR1 SEQ ID NO: 12, CDR2 SEQ ID NO: 13, CDR R3 sequence number:14.

[0184] In some embodiments, the method, dosage or administration regimen of treatment comprises administering to the patient a therapeutically effective amount of an anti-α4β7 antibody. For example, the anti-α4β7 antibody-reactive HAHA reaction is reduced. The occurrence of HAHA, as measured by antibodies, enhances the clearance of anti-α4β7 antibodies. For example, the serum concentration of anti-α4β7 antibodies can be reduced, for example, It reduces the number of anti-α4β7 antibodies bound to α4β7 integrin, reducing the efficacy of treatment. In some embodiments, an induction regimen followed by a maintenance regimen may be used to prevent HAHA. In some embodiments, a break may be taken between the induction and maintenance regimens. In some embodiments, the induction regimen involves administering multiple doses of an anti-α4β7 antibody to the patient. When starting treatment with anti-α4β7 antibodies to prevent HAHA, A high initial dose, e.g., at least 1.5 mg / kg, at least 2 mg / kg, at least 2.5mg / kg, at least 3mg / kg, at least 5mg / kg, at least At least 8 mg / kg, at least 10 mg / kg or about 2 to about 6 mg / kg, or frequent initial doses Administration, for example, about once per week, about once every two weeks, or about once every three weeks The patient can be treated with standard doses. In some embodiments, the treatment method includes administering at least at least 30%, at least 40%, at least 50%, at least 60%, at least 7 0%, at least 80%, at least 90% or at least 95% as HAHA negative In other embodiments, the treatment method comprises at least 6 weeks, at least 10 weeks, or at least 15 weeks, at least 6 months, at least 1 year, at least 2 years, or The patient remains HAHA negative for the duration of the therapy. or at least 30%, at least 40%, or at least 5% of patients experiencing HAHA 0% or at least 60% maintain a low titer of anti-α4β7 antibodies, e.g., ≦125. In embodiments, the method of treatment includes treating at least 70% of patients with an anti-α4β7 antibody. Maintain HAHA negative status for at least 12 weeks after initiation of treatment.

[0185] The formulations may be administered to an individual (e.g., a human) alone or in combination with another agent. The formulation may be administered prior to, together with, or after administration of the additional agent. In one embodiment, more than one formulation that inhibits the binding of α4β7 integrin to its ligands is administered. In such embodiments, the agent, e.g., anti-MAdCAM or anti-VCAM-1 A monoclonal antibody, such as a monoclonal antibody, can be administered. So, what if the additional agent inhibits leukocyte binding to endothelial ligands in a pathway different from the α4β7 pathway? Such agents inhibit chemokine (CC motif) receptor 9 (CCR9)-expressing The expression of thymic chemokines (TECK or CCL25) or LFA-1 in lymphocytes It can inhibit binding to agents that inhibit binding to intracellular adhesion molecules (ICAMs). For example, anti-TECK antibodies or anti-CCR9 antibodies or PCT Publication WO03 / 099773 or Small molecule CCR9 inhibitors, such as those disclosed in WO04 / 046092, or anti-IC AM-1 antibodies or oligonucleotides that interfere with the expression of ICAM may be added to the formulations of the invention. In yet another embodiment, an additional active ingredient (e.g., sulfonamide, Anti-inflammatory drugs containing asalazine, azathioprine, 6-mercaptopurine, and 5-aminosalicylic acid anti-inflammatory compounds such as steroids, other nonsteroidal anti-inflammatory compounds, steroidal anti-inflammatory compounds Compounds, or antibiotics commonly administered for the control of IBD (e.g., ciprofloxacin, or other biological agents (e.g., TNFα antagonists) in the preparations of the present invention. It can be administered in combination with a drug.

[0186] In embodiments, the doses of the co-administered agents are adjusted according to the dosage regimen of the anti-α4β7 antibody-containing formulation. For example, the dose can be reduced over time after the start of treatment with anti-α4β7 antibody preparations. Treated with steroids (e.g., prednisone, prednisolone) at or before the onset of Patients who had received anti-α4β7 antibody therapy were recommended to receive steroid therapy starting as early as 6 weeks after the initial treatment. The patient will be placed on a tapered regimen. The steroid dose will be reduced by 4-8 times the initial dose. Approximately 25% of patients were treated within 8 weeks, and 5% were treated within 8 to 12 weeks of tapering the dose of anti-α4β7 antibody preparations. 0% and 75% in about 12-16 weeks. Steroid therapy can be eliminated by approximately 16-24 weeks of treatment with the formulation. Therefore, it is recommended to use 6-mercaptopurine or similar at the start of or before treatment with anti-α4β7 antibody preparations. Patients who were treated with such anti-inflammatory compounds were, as mentioned above, Receive a regimen of decreasing doses of anti-inflammatory compounds similar to the dose-tapering regimen used in become.

[0187] In one embodiment, the method comprises administering to a patient an effective amount of a formulation of the invention subcutaneously or intramuscularly. In another embodiment, the formulation may be prepared for self-administration.

[0188] If the formulation is in a solid, e.g., dry, state, the process of administration may convert the formulation to a liquid state. In one embodiment, the method includes administering the compound to the subject by injection, e.g., intravenously, intramuscularly, or subcutaneously. For use in injections, the dry formulation can be reconstituted with a liquid, for example as described above. In another embodiment, the solid or dry formulation can be, for example, a patch, cream, Administration can be topical via aerosol or suppository.

[0189] The present invention relates to a method for the detection of MAdCAM-1-expressing cells in tissues expressing the molecule MAdCAM (e.g., MAdCAM-1). The present invention also relates to a method for treating a disease associated with blood cell infiltration. In an embodiment, the disease is a graft. In some embodiments, the disease is a disease caused by the molecule MAdCAM (e.g., MAdCAM-associated inflammatory disease). The α4β7 integrin-expressing cytoplasmic leukocytes (CL-1) expressing endothelium associated with the gut In another embodiment, the present invention relates to a disease associated with leukocyte infiltration of tissues as a result of binding of blood cells. The disorder may be gastritis (e.g., neutrophilic or autoimmune gastritis), pancreatitis, or insulin-dependent In yet another embodiment, the disease is cholecystitis, cholangitis, or pericholecystitis. do.

[0190] The present invention also relates to a method of treating inflammatory bowel disease in a patient. The method includes subcutaneously administering to a patient an effective amount of an anti-α4β7 antibody formulation of the present invention. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. Inflammatory bowel disease is a condition that can include celiac disease, seronegative arthropathy-associated enteropathy, microscopic or collagenous colitis, gastroenteritis (eg, neutrophilic gastroenteritis), or pouchitis.

[0191] In some embodiments, treatment with an anti-α4β7 antibody alters the ratio of CD4:CD8 lymphocytes. No. CD4:CD8 ratios are measured in blood, lymph node aspirates, and cerebrospinal fluid (CSF). CSF CD4 in healthy individuals + :CD8 + The ratio of lymphocytes to 1 or higher (Svenningsson et al., J. Neuroimmunol. 1995;63:39-46; Svenningsson et al., Ann Neurol. 1993; 34:155-161). Immunomodulatory agents are used to reduce the CD4:CD8 ratio to less than 1. It can be changed.

[0192] manufactured goods In another aspect, the invention provides a pharmaceutical formulation of the invention containing instructions for use. An article of manufacture. The article of manufacture includes a container. Suitable containers include, for example, bottles, vials ( For example, dual chamber vials, vials of liquid preparations with or without needles, vials with or without needles vials of solid formulations with or without vials of reconstitution fluid without needles), syringes (e.g. Examples of suitable containers include dual chamber syringes, pre-filled syringes, autoinjectors), cartridges, and test tubes. The containers can be formed from a variety of materials, such as, for example, glass, metal, or plastic. The container holds the formulation and a label thereon or associated therewith, and the container can indicate instructions for use. In another embodiment, the formulation is prepared for self-administration and / or can contain instructions for self-administration. In one aspect, the container holding the formulation can be a single-use vial. In another aspect, the container holding the formulation can be a multi-use vial, which allows for repeated administration of the formulation (e.g., 2-6 administrations) using more than one portion of the reconstituted formulation. The article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles, syringes, and package inserts with instructions for use as mentioned in the previous section.

[0193] In one embodiment, the article of manufacture is a syringe with a needle. The needle gauge is 25G, 26G, Thin-wall needles, e.g., 19G or 23G or larger, can be used for viscosity control. In one embodiment, the gauge of the needle is 27G or more. The length of the needle may be any length suitable for subcutaneous administration, and may be 1 / 2 inch, about 5 / 8 inch or In some embodiments, the syringe may be a pre-filled syringe. It is Ji.

[0194] Pre-filled Syringe Product Development In some embodiments, a pre-filled syringe (PFS) (e.g., for subcutaneous or intramuscular delivery) is used. for use in administering formulations for intravenous delivery) There are several product attributes that are desirable for the 4β7 antibody. It is useful to balance some of the attributes. For example, if a small injection volume is desired, In such cases, a high protein concentration of the formulation may be preferred. , high rates of impurity formation (e.g., aggregated impurities seeping out of the syringe into the drug product) , the manual force required to operate the syringe can be high. The small size of the needle can require a large force to operate the syringe. Product stability is determined by both formulation and needle parameters such as protein concentration, pH and needle diameter. Understanding how the quality and performance of protein products (e.g. Anti-α4β7 antibodies in pre-filled syringes) to help develop.

[0195] In one embodiment, a protein product (e.g., For example, the method for developing a 4β7 antibody (anti-α4β7 antibody) involves the use of syringe parameters and formulation parameters. Including varying together, e.g., in a coordinated manner or simultaneously. If this continues to change, this will be from a protein product in a pre-filled syringe. This can provide a better understanding of the product stability and range of product performance that can be expected from do.

[0196] The development of pre-filled syringe products (e.g., anti-α4β7 antibodies) may become a reality at some point. Relevant to the understanding that there is a liquid formulation that comes into contact with some components of the previously filled syringe. (FIG. 15). For example, the formulation may come into contact with a syringe barrel, which may be made of glass (e.g., type I). borosilicate glass) or plastic (e.g., cyclic olefin polymer (COP) , cyclic olefin copolymer (COC), polypropylene or polytetrafluoroethylene The formulation may be constructed from a syringe, a plunger and / or a tip cap. They may be made of elastomers (e.g., the same or different materials (e.g., polyester). Plastics or rubbers (natural, synthetic, The formulation may be made of a plastic such as butyl or silicone. The syringe may be contacted with a lubricant that is added to the syringe barrel to facilitate movement. Lubricants include, for example, The needle may be silicone oil, mineral oil, or glycerin. So, a metal alloy needle (for example, a stainless steel needle and an adhesive that holds the needle in place) Possibly. Some considerations for protein products in pre-filled syringes are The liquid protein solution will remain in one or more of these syringe components throughout the product's shelf life. Both the formulation and the syringe components are designed to stabilize the protein. This can have a qualitative impact.

[0197] Formulation parameters that can affect the stability of prefilled syringe products These include protein concentration, pH, type of buffer, ionic strength, type and amount of stabilizer. Examples of stabilizers for protein formulations include, for example, ionic salts, These include polysaccharides, amino acids, antioxidants, chelating agents, and surfactants as described in the previous section. Can be obtained.

[0198] Syringe components that can affect the stability of pre-filled syringe products These include, for example, lubricants, plunger and end cap composition, and impurities. The amount of lubricant (e.g., silicone oil in syringes) affects the stability of the product. It can affect the oxygen permeability of these components and can affect the oxygen permeability of protein products (e.g., A plunger and a device capable of introducing exudates from these components of the α4β7 antibody formulation The composition of the tip cap and the encapsulation agent may also affect the stability of the product. Additional syringe parameters to obtain include (e.g., barrel (e.g., glass barrel) and / or is a contaminant that can leach from a needle (e.g., a stainless steel needle) into the product formulation. The type and / or amount of impurities (e.g., heavy metals (e.g., tungsten)) (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: (see also 564-572).

[0199] Pre-filled syringes can be used for injection manually or with an auto-injector device. Functionality testing of pre-filled syringes includes measuring the pull-out force, the force required to initiate the plunger movement, and the slippage force, the force required to inject the contents of the syringe at a constant rate. The mechanical performance of pre-filled syringes can depend on several parameters of the formulation and the syringe, such as the viscosity of the formulation and the amount of lubricant (e.g., silicone oil) in the syringe.

[0200] Some attributes of protein products in pre-filled syringes and their product characteristics Formulation and syringe factors that can affect quality are shown in Table 1. Many product attributes are , can be a complex function of several parameters of the formulation and the syringe. For example, viscosity is It depends on several factors of the formulation, such as protein concentration, stabilizer concentration, and pH. However, the sliding force of the syringe is a function of the viscosity of the formulation.

[0201] [Table 1]

[0202] In one embodiment, an interfacial agent such as, for example, polysorbate 20 or polysorbate 80 is used. The active agent can be added to the protein formulation in a pre-filled syringe (e.g. For example, tamper-evident properties at liquid / air and / or liquid / lubricant (e.g., silicone oil) interfaces. (To prevent the adsorption and denaturation of protein molecules on the surface) is one of the mechanisms for nucleation of subvisible and visible proteinaceous particles. Therefore, adding surfactant to a pre-filled syringe may result in the pre-filled syringe being The formation of sub-visible and visible particles in Linde's products can be reduced. In one embodiment, a small amount of a surfactant can emulsify the lubricant (e.g., silicone). Corn oil creates droplets in solution that act as both invisible and visible lubricants. For example, reducing the formation of oil droplets in silicone oils (Ludwig et al., supra). In some cases, the formulation should be carefully considered due to the potential adverse effects of large amounts of surfactant on protein formulations. Reduce the amount of surfactants in the solution as much as possible. Peroxide impurities present in polysorbates , which can result in high protein oxidation (Wang and Wang J. Pharm. Sci. 91:225 2-2264 (2002). A large amount of surfactant was found to remove a significant amount of silicone oil from the walls of the syringe. This can result in increased functional slippage over a useful period of time. Product development studies have evaluated the effect of various surfactant levels on both product stability and syringe performance. The study should be designed to examine the effect of

[0203] Complexity between formulation and syringe parameters in protein / PFS systems Such interactions can be achieved using a quality by design (QbD) or design of experiments (DOE) approach. It is suitable for testing these systems. Studies can be designed that combine these data to gain a better understanding of these complex systems. This results in a comprehensive approach to the development of a prefilled syringe product. Input to review examples of experimental designs and analytical tests employed for filled syringe products Examples of parameters and levels are given. Depending on the type of experimental design used for QbD testing, The number of experiments ranged from 9 for a screening design to a full factorial design (all possible combinations). The more experiments you run, the more correlations between product parameters you can resolve. The number of interactions is large. Software designed for this analysis, e.g., JMP (registered Statistical Discovery Software (Cary, North Carolina) can aid in QbD testing This analysis will explore how formulation parameters and syringe parameters interact to determine the product Generates a quantitative understanding of how factors affect a characteristic.

[0204] [Table 2]

[0205] An example of a predictive model that can be derived from the experiments of the examples shown in Table 2 is provided below, where: C n is a numeric constant.

[0206] Formation of soluble aggregates over time = C0 + C1 [protein concentration] + C2 [protein concentration] ] 2 + C3 [pH] + C4 [Surfactant concentration] + C5 [Amount of lubricant]

[0207] Many parameters of the syringe can affect the stability and performance of the product. In some embodiments, acceptable tolerance in syringe parameters is consistent with product stability and performance. This includes a characterization of how lubricants in syringes affect the performance of the syringe. For example, the amount of silicone oil can vary from 50 to 100% from syringe to syringe. This variation in can affect several product characteristics, as shown in Table 1. The inner diameter may vary from syringe to syringe, which affects the injection force. The inner diameter of the needle varies from lot to lot and from manufacturer to manufacturer, which affects the injection force. To examine how syringe parameters affect performance, Qb Using the D approach, it is possible to determine how acceptable tolerances in syringe parameters affect product performance. This allows us to obtain predictive models that can be used to estimate how The predictive models obtained using the QbD approach can be used to refine manufacturing processes to meet desired product attributes. Agent and syringe parameters can be selected to predict product stability and performance.

[0208] Pre-filled syringes deliver silicone emulsion or tang to protein formulations. Examples of silicones that may be present in the pre-filled syringes are: The amount ranges from about 0.3 mg to about 0.8 mg. In one embodiment, the pre-filled syrup is The amount of silicone that may be present in the syringe is about 0.3 mg, about 0.4 mg, about 0.5 mg, about In another embodiment, the viscosity of the formulation is 2 The range will be 2 to 60 cP, which will produce an injection force of 5 N to 80 N at a speed of 0.1 00 mm / min. In yet another embodiment, the viscosity of the formulation is such that the injection force is 10N to 40N at a speed of 200 mm / min. The resulting range will be 4 to 27 cP.

[0209] The present invention will be more fully understood by reference to the following examples. While these are not intended to limit the scope of the present invention, they should not be construed as limiting the scope of the present invention. All citations by Xue are incorporated herein by reference.

[0210] Protocol for making the formulation The anti-α4β7 antibody solution was diafiltered in a tangential flow filtration system to remove citrate, histidine, and The mixture is then diluted with arginine to a specific concentration and pooled, and diluted with citrate, histidine, and arginine. Mix with a solution of polysorbate 80 in arginine buffer. The solution is stored in a bottle at -70°C. The solution is then thawed and filtered twice through a 0.2 μm filter. Approximately 1.0 mL is loaded into a sterile syringe and inserted with a sterile plunger (stopper The formulation is stored and the final drug product is shipped in syringes at 2-8°C. EXAMPLES

[0211] Example 1: Preparation of formulation factor Excipient concentration Aggregate formation in antibody formulations was investigated. Protein concentration, pH and surfactant: The SEC aggregate model was developed from experimental data examining the molar ratio of proteins. In the pH range, the formation of aggregates was suppressed at polysorbate 80 to protein molar ratios of 0.7 to 1. 0.5 (Figure 6). In general, PS80 to protein ratios higher than 1.5 The rate of aggregate formation increases with increasing pH (Figure 7).

[0212] Experiments were carried out to investigate the formation of SEC aggregates in the presence of air. The formulation was placed in a borosilicate vial and sealed with an elastomeric stopper with air headspace. An identical set of formulations was made and the air headspace was replaced with argon. The samples were allowed to stabilize at 40°C for 2 weeks. All samples with air headspace were stored in Argon. This resulted in a large amount of aggregates at the end of the experiment compared to the same formulation with headspace.

[0213] [Table 3]

[0214] Based on these experiments, SEC aggregates may be formed by oxidation or by disulfide bonds. The addition of antioxidants and / or chelating agents was investigated. Polysorbate 80:protein molar ratio of 1.5 was used with 40 mM histidine, 90 mM A formulation was made containing 2 M arginine and 160 mg / mL protein. 5 mM citrate, 5 mM citrate, 5 mM EDTA, 25 mM cysteine ​​or All three added excipients suppressed the formation of aggregates ( Figure 8). The addition of antioxidants and / or chelating agents improved performance in the following order: citrate > EDTA > silsesquioxane. The 5 mM or 25 mM citrate was ranked as staining compared to the control formulation. This suppressed the formation of SEC aggregates.

[0215] pH, protein concentration, citrate concentration, histidine concentration and polysorbate 80 and ta An experiment was conducted to determine the effect of the molar ratio of protein on the pH. Protein concentrations were varied from 60 to 160 mg / mL, and citrate concentrations were varied from 0 to 25 mM. The histidine concentration was varied from 25 to 50 mM, and polysorbate 80 and protein The molar ratio of was varied from 0.7 to 1.5. The formulations were placed in a 1 mL 27G1 / 2” syringe. (0.55±0.2 mg silicone). All formulations contained approximately 125 mM alginate. It contained nin.

[0216] The stability was examined at 40°C for 2 weeks using CEX and SEC. The results (Figure 9 and Table 4) ) showed that increasing protein concentration increased the rate of aggregate formation, whereas 2 The presence of 5 mM citrate shows a reduction in aggregate formation. While the amount of monomer remains essentially unchanged up to 4 months, the amount of monomer increases with increasing aggregate formation at 25°C and 40°C. The results show an opposite trend (Table 5).

[0217] Another set of formulations was performed in the presence of 40–63 mM citrate at 40°C, 25°C, and 5°C. However, the rate of SEC aggregate formation in the absence of histidine was examined. The rate of aggregate formation in the formulation with histidine was slightly higher at 40°C. At 5°C, however, there was no significant increase in aggregates in the formulation with citrate but without histidine. The formation rate was comparable to that of formulations containing citrate and histidine (Table 6). However, the amount of monomer remained essentially unchanged up to 24 months (Table 7).

[0218] [Table 4]

[0219] [Table 5]

[0220] [Table 6]

[0221] [Table 7]

[0222] pH Several pH experiments were carried out to determine the effect of pH on the decomposition of CEX at 5°C. The izumab antibody formulation consisted of 160 mg / mL anti-α4β7 antibody, 125 mM arginine, 50 The mixture was composed of 25 mM histidine and 25 mM citrate. Several different pH levels were examined for stability: 6.3, 6.5, 6.7, and 6.9. . The CEX model at 40°C (Figure 10) shows that pH has the greatest effect on CEX decomposition. The pH of the histidine-containing formulations decreased with increasing temperature, whereas that of the citric acid-containing formulations decreased with increasing temperature. The pH of the salt formulations was shown to be unaffected by temperature (Figure 11). Histidine / Citrate The formulation was pH 6.8 after 1 week at 40°C and pH 6.3-6.5 after 6 months at 25°C. It was confirmed that the compound has good stability at pH 6.3-6.5 after 6 months at 5°C. Based on additional testing, the stability of the formulation was demonstrated at 25°C and 5°C in the pH range of 6.2 to 6.9. The results were similar in terms of temperature (Tables 8 and 9).

[0223] [Table 8]

[0224] [Table 9]

[0225] Example 2 stability Four different anti-α4β7 antibody formulations were examined for stability over a 12-month course. Formulations with a pH of 6.0-6.2 were approximately 10% lower than those with a pH of 6.3-6.4. The formulations with pH values ​​of 6.3 to 6.4 showed 1 to 2% less of the major species (Figure 12). showed less than 1% change in basic or major species.

[0226] Stability of 10 different anti-α4β7 antibodies by SEC over a 12-month period The agents tested were (Table 10). The 160 mg / mL formulation had a 0.1-0.2% change in aggregates after one year, whereas the 160 mg / mL formulation had a 0.1-0.2% change in aggregates after one year. Formulations containing protein and 25 mM citrate showed 0.2-0.3% The 60, 110 or 160 mg / mL doses without citrate had an increase in aggregates. For formulations with, there was an increase in aggregates of 0.4-0.6%.

[0227] [Table 10]

[0228] Example 3 viscosity The injection force required to administer a pharmaceutical formulation is related to the viscosity of the formulation. Various concentrations of protein, arginine, histidine, citrate, sucrose and polysaccharides were Formulations with Resorbate 80 were prepared. The viscosities of these formulations were examined. Ln (viscosity) A statistical model was developed for the viscosity of the protein. The model showed that the viscosity is mainly affected by the protein concentration and pH. Sucrose, histidine and arginine also had a light effect on viscosity (Figure 13). Some protein formulations are formulated with sodium chloride. However, the effect of sodium chloride on viscosity is not and formulation dependent.

[0229] 140mg / ml vedolizumab, 125mM arginine, 25mM histidine, Polysorbate 80 at 25 mM citrate and a molar ratio of polysorbate 80 to protein of 1.5 Sodium chloride was added to the formulation, which contained bait 80 and had a pH of 6.4. It had no effect on the viscosity of the agent.

[0230] The effect of viscosity on injection force for the various syringes examined is shown in Figures 16A and 16B.

[0231] Example 4 method Cation Exchange Chromatography (CEX) Phosphate / sodium chloride using a weak cation exchange column in high performance liquid chromatography A gradient of 1000 mAbs was used to separate the charged species in the anti-α4β7 antibody formulation and measure the charge composition of the antibody species. The acidic isoform elutes before the major isoform and after the major isoform. The basic isoform elutes.

[0232] Stability data for vedolizumab formulations generated using the CEX assay showed that the major isoforms The results showed that the % of the groups exceeded 55.0%.

[0233] Capillary isoelectric focusing (cIEF) iCE280 full-column detection cIEF system (Convergence, Toronto, Ontario) cIEF was performed using a ELISA kit (Gentile Biosciences). Therefore, the combination of ampholytes can be selected as recommended and is commercially available. Useful combinations include 3-10 and 5-8 PHARMALYTE® (New Zealand) The company is GE Healthcare, Piscataway, Jersey.

[0234] Stability data for vedolizumab formulations generated using the cIEF assay showed that the major isofluranes The percentage of form is about 53%, acidic species is about 42%, and basic species is about 5%. showed.

[0235] Size Exclusion Chromatography (SEC) Analytical SEC column (Tosoh Bios, King of Prussia, PA) SEC was performed using a column chromatography column (Cience, LLC). The mobile phase was a phosphate buffered saline solution. and the absorbance is monitored at 280 nm.

[0236] Stability data for vedolizumab formulations generated using SEC assay showed that the % monomer was 9. 9.0%, % of aggregates is <0.5%, and % of low molecular weight material is <1.0% It was shown that.

[0237] SDS-PAGE assay Invitrogen (CaCl) was used at 4-20% under reducing conditions and 4-12% under non-reducing conditions. SDS-PAGE was performed using Tris / glycine gels (Carlsbad, California). The reconstituted antibody formulation is diluted with liquid formulation buffer and then diluted with 10% 2-mercapto with ethanol (reduced sample buffer) or without 2-mercaptoethanol (non-reduced Sample Buffer) 1: in Tris / Glycine SDS sample buffer (2X, Invitrogen) The samples were briefly heated and contrasted with molecular weight markers (Invitrogen). Colloidal Coomassie Blue (Invitrogen) was added according to the manufacturer's instructions. Stain the gel with 100 mM NaCl. Analyze the protein bands by densitometry and reduce the Gels specify % heavy and light chains, non-reduced gels specify % IgG.

[0238] Bond Efficiency HuT78 cells (human T-cell lymphoma cells, American Type Culture Collection, Manassas, Virginia. After incubation on ice, the cells are contacted with serial dilutions of the primary test antibody. After washing, the cells are treated with a fluorescently labeled secondary antibody. After further washing, the cells are fixed and then Itometry (Becton Dickinson, Franklin Lakes, New Jersey) The cells are then suspended in FACS reagent for analysis by FACS (Fascinson). See also issue 851.

[0239] Moisture by Karl Fischer Formulations are titrated with methanol for coulometric Karl Fischer water determination.

[0240] Example 5 The influence of silicone from pre-filled syringes containing anti-α4β7 antibody formulations Contains L-histidine, L-arginine hydrochloride, citrate and polysorbate 80. A subcutaneous preparation containing 60 to 160 mg of anti-α4β7 antibody protein in a buffer solution containing The study examines the effect of silicones on the stability of protein formulations and on the properties of containers / closures. Use 0.5 mL portions.

[0241] Protein concentration, molar ratio of polysorbate 80 to protein, and amount of the solution sprayed into the syringe The parameters including the amount of silicone are examined. The ranges of each of the input parameters are shown in Table 11. show.

[0242] [Table 11]

[0243] Use an experimental design to determine the set of formulations to be examined. A reasonable number of formulations is 6-8 formulations. Examples of formulations to be investigated are shown in Table 12.

[0244] [Table 12]

[0245] Some controls may be included in the set of formulations and tested at a few selected time points.

[0246] At several different temperatures (e.g., 5°C, 25°C / 60%RH, 40°C / 75%RH) These formulations were allowed to stand and were then assayed at various time points (e.g., 0 weeks, 1 week, 2 weeks, 4 weeks, 8 weeks, 1 week, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, The rats were removed for testing at 0 weeks, 12 weeks, 6 months, and 12 months. Find out in 12 months.

[0247] Tests performed at each stability time include SEC, CEX, Instron, MFI, One syringe was examined with an Instron to determine the amount of material released. The silicone was analyzed by SEC, CEX, injection force measurement, and microflow imaging (MFI). Used for quantitative determination.

[0248] Example 6 Analysis of the components of pre-filled syringes with anti-α4β7 antibody formulations This test is carried out on various syringe manufacturers, plungers (stoppers), elastomers, How does the amount of PS80 in the material and formulation affect the mechanical properties of the system and the stability of the formulation? We investigated how this affects

[0249] Three different syringe manufacturers, two different plunger (stopper) materials, A design of experiment was created to examine two different PS80 to protein molar ratios. The solution contains 170 mg / mL protein, 125 mM arginine, and 50 mM histidine. The pre-filled 100 mM citrate and pH were kept constant at 25 mM citrate and 6.5. The syringe needle size is 27G. 1 / 2' or 29G 1 The thin wall thickness was 2 / 2". Details are provided in Table 15.

[0250] The experimental design inputs for the moving parts of the experiment are shown below in Table 13, while the constant parts are shown in Table 14. An experimental design was created using the inputs shown in Table 13.

[0251] The list of experiments is shown in Table 10.

[0252] [Table 13]

[0253] [Table 14]

[0254] [Table 15]

[0255] Concentrated preparation, anti-α4β7 antibody preparation, with polysorbate 80 added to make it 170 mg / mL The composition of the starting formulation is shown below in Table 16.

[0256] [Table 16]

[0257] To dilute the substance to the desired formulation, 25 mM citrate, 50 mM histidine, Make a stock solution of PS80 in 125 mM arginine, pH 6.48.

[0258] [Table 17]

[0259] The formulation dilution scheme is detailed in Table 18.

[0260] [Table 18]

[0261] The compounding should be done based on the dilution scheme and the starting formulation should be weighed out, but other stock dilutions should be The liquid can be pipetted volumetrically. The formulation is filtered. 0.5 mL of the formulation is Dispense into as many 1 mL long syringes as possible. Press with a 2-4 mm bubble. For each time point, one syringe is reserved with the needle facing down and the other is One syringe should be stored horizontally, and the extra syringe should be stored needle-side down.

[0262] Examine the syringes at 5, 25 and 40°C for 2 weeks and 1 month. Analytical tests (appearance, ink, etc.) stron, pH, osmolality, density, viscosity, SEC, CEX and Brightwell) first. Then again at 25°C and 40°C for the second week and at 25°C for the fourth week.

[0263] Example 7 Subcutaneous container closure for use with a 27G thin-walled needle syringe prefilled with anti-α4β7 antibody formulation Analysis of the stoppage The study consisted of different syringe models with 27G thin-walled needles and different plungers ( How does the make and model of the topper affect the mechanical properties of the system and the stability of the formulation over time? Investigate how this affects

[0264] This test is conducted by the manufacturer of the syringe and the plunger ( Anti-α4β7 antibody subcutaneous solution in a pre-filled syringe with stopper (model To investigate how the stability of the formulation and the mechanical properties of the syringe are affected. Data generated from the container / closure components for liquid subcutaneous anti-α4β7 antibody formulations It can be determined.

[0265] The experimental design inputs are shown below in Table 19, with the constants given in Table 20. The experimental design was created using

[0266] A list of the experiments performed is given in Table 21.

[0267] [Table 19]

[0268] [Table 20]

[0269] [Table 21]

[0270] The concentrated anti-α4β7 antibody preparation was diluted with polysorbate 80 to 160 mg / mL. The composition of the starting formulation is shown below in Table 22.

[0271] [Table 22]

[0272] To dilute the substance to the desired formulation, add 25 mM citrate, 50 mM histidine, Make a stock solution of PS80 in 125 mM arginine and 125 mM arginine, pH 6.3. .

[0273] [Table 23]

[0274] The dilution scheme for the formulation is detailed in Table 24.

[0275] [Table 24]

[0276] The compounding should be done based on the dilution scheme and the starting formulation should be weighed out, but other stock dilutions should be The liquid can be pipetted volumetrically. The formulation is filtered. 0.5 mL of the formulation is Dispense into as many 1 mL long syringes as possible. Press with a 2-4 mm bubble. Tighten the syringe by twisting the needle. For each time point, one syringe is saved with the needle facing down (horizontal). position).

[0277] 5℃, 25℃ / 60%RH and 40℃ / 75%RH for 1 month, 3 months, 6 months, 9 months ( (Optional), and syringes will be examined at 12, 18, and 24 months.

[0278] 1, 3, 6, 9, 12, 18, 24 months (5℃), 1, 3, 6, 9, 12, 18 months ( Liquid formulations at 25°C for 1, 3, 6, 9, and 12 months (40°C) and 1 and 3 months (40°C) Analytical investigations (concentration, osmolality, pH, Instron, MFI, SEC, and / or CE X).

[0279] Example 8 Analysis of subcutaneous anti-α4β7 antibody formulations in plastic pre-filled syringes Use of a plastic syringe as a container / closure system for a subcutaneous anti-α4β7 antibody formulation The study is being launched to study the use of pre-filled plastic candidate syringes. The stability of a representative anti-α4β7 antibody subcutaneous formulation in the sham was examined. The data indicate the applicability of plastic syringes for use with liquid subcutaneous anti-α4β7 antibody formulations. It helps to decide.

[0280] The stability test samples are prepared as follows. The stability test is performed at 40°C / 75% RH, The test is carried out under storage conditions of 25°C / 60% RH and 5°C.

[0281] The liquid subcutaneous anti-α4β7 antibody formulation shown in Table 26 was used together with the two types of plastic A plastic syringe and one glass syringe (control) were examined. Table 27 shows the results of the experiment. The details of each set of samples are shown.

[0282] [Table 25]

[0283] [Table 26]

[0284] [Table 27]

[0285] The liquid subcutaneous anti-α4β7 antibody formulation prepared previously will be used in this study. The formulation will be filtered. Sample the filtered solution for quality testing as a "before filling" sample (appearance, MFI, DLS). Dispense 0.5 mL of the formulation into a 1 mL plastic syringe. Vacuum clamp Tighten the syringe with a . Store the syringe with the needle facing down.

[0286] An initial check is performed to measure pH, osmolality, density, viscosity and protein concentration. Analysis test (appearance, SEC (aggregate, monomer, LMW), CEX (acid, main, basic), (strength, MFI, DLS and / or weight) after 1 week at 40°C, after 2 weeks at 40°C, After 1 month at 25 and 40°C, after 3 months at 5 and 25°C, after 6 months at 5 and 25°C, After 9 months at 5 and 25°C, and after 12 months at 5 and 25°C.

[0287] Samples were collected at 1, 3, 6, 9, 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

[0288] Appearance at 5°C and 25°C at various time points which may include 0, 1, 3, 6 and 12 months The samples were analyzed for injection force, SEC, CEX and microflow imaging. The stability of the formulations as measured by CEX was similar to that discussed in Examples 1 and 2. For the injection test, the slip force was measured (Table 28). The statistical model was The only significant factor affecting the results was the syringe manufacturer, and A was found to be more likely to The slipperiness of the 12-month test at 5°C and the 6-month test at 25°C was higher than that of the 12-month test at 25°C (Figure 17). The change in the sliding force of the syringe over the month was less than 10 N, mostly less than 5 N.

[0289] [Table 28]

[0290] Example 10 A pre-filled syringe used in a 27G thin-walled needle syringe filled with an anti-α4β7 antibody formulation. Analysis of the components of Lingzi This test was carried out on various manufacturers of syringes with 27G thin-wall needles and plungers (stoppers The manufacturer and elastomeric material of the prefilled syringe system The effect of chlorine on the stability of the formulation over time was investigated.

[0291] 27G 1 160mg / mL protein at pH 6.5 with 2" thin wall needle, 125m M arginine, 50 mM histidine, 25 mM citrate, 0.2% PS80 Three different syringe manufacturers and four different plungers ( All samples created and examined are listed in Table 29.

[0292] [Table 29]

[0293] at 5°C, 25°C and 40°C at various time points which may include 0, 1, 3, 6 and 12 months. The samples were analyzed for appearance, injection force, SEC, CEX and microflow imaging. The stability of the formulations as measured by SEC and CEX was discussed in Examples 1 and 2. The injection test was similar to that of the previous one. The ejection and slippage forces were measured. The results are shown in Table 30.

[0294] [Table 30]

[0295] The statistical model showed that syringe manufacturers A and C had similar, but lower, slippage forces than manufacturer B. However, plunger (stopper) E was slightly weaker than the other plungers (stoppers). It was shown to have high sliding force.

[0296] In general, the initial pull-out forces were similar among the samples examined.

[0297] The slip force did not change significantly over 12 months at 5°C, 25°C, and 40°C. However, the withdrawal force of the syringe with plunger (stopper) F was 25°C and 4 It increased to 12 months at 0°C.

[0298] Example 11 Analysis of anti-α4β7 antibody formulations in pre-filled syringes The study included various levels of protein concentration, polysorbate 80 concentration, and citrate. How concentration and pH affect anti-α4β7 antibody formulations in pre-filled syringes Investigate what impact this has.

[0299] Protein concentration (60–160 mg / mL, pH (6.0–6.3), polysorbate 80:2 molar ratio of protein (0.723-1.5) and citrate (0-25 mM) Create part of the experimental design in JMP by implementing the partial level. These formulations are histidine-concentrated The concentration of arginine (125 mM) and the concentration of glycerol (50 mM) were constant (Preparations 1 to 8). Variants of these formulations with 1 mM histidine are added (formulations 9-10).

[0300] An additional set of formulations was developed to eliminate histidine and use only citrate as the buffer. The formulations that have the same effect are examined (formulations 11 to 16). The input level is shown for all the formulations examined. The constants used for all formulations are shown in Table 32.

[0301] [Table 31]

[0302] [Table 32]

[0303] [Table 33]

[0304] Each formulation contained an anti-α4β7 antibody and was diluted in various excipient stock solutions. To achieve a reasonable dilution volume, the anti-α4β7 antibody stock used was The stock solution is shown in Table 34. Two different TFF procedures were performed to obtain formulations TFF1 and 2. A portion of TFF1 is used for dialysis to yield the formulation labeled "Dialysate."

[0305] [Table 34]

[0306] To dilute the material to the desired formulation, aqueous solutions of each excipient at the concentrations specified in Table 35 are used. Make.

[0307] [Table 35]

[0308] The dilution schemes for the formulations are detailed in Tables 36 and 37.

[0309] [Table 36]

[0310] [Table 37]

[0311] Blends are made based on the dilution scheme, starting formulations are weighed, while other stock solutions are mixed by volume. Pipette analytically. Filter the formulation. Pipet 0.5 mL of the formulation into as much 1 mL as possible. Dispense into a long syringe. Tighten the syringe with a tightening machine. The syringe is placed with the needle facing down. Save.

[0312] The liquid formulation was initially incubated at 40°C for 1 week, at 40°C for 2 weeks, and at 25°C and 40°C for 1 month. at 5℃ and 25℃ for 2 months, at 5℃ and 25℃ for 3 months, at 5℃ and 25℃ for 6 months At 5°C and 25°C for 9 months, at 5°C and 25°C for 12 months, analytically (appearance, pH, Examine samples using a variety of techniques (osmolarity, density, DLS, SEC, CEX and / or Brightwell).

[0313] Extractions are also performed for specific formulations according to Table 38.

[0314] [Table 38]

[0315] Example 12 in either glass syringes or two different COP plastic syringes. The stability of the 160mg / ml protein, 50mM histidine, 25mM A formulation containing citrate, 125 mM arginine at pH 6.5 was examined. After that, the aggregate and monomer amounts were measured at 5°C or 25°C using plastic and glass syringes. It was comparable.

[0316] [Table 39]

[0317] Example 13: Bioavailability of vedolizumab administered by subcutaneous and intramuscular injection Bioavailability of vedolizumab administered by subcutaneous and intramuscular injection in healthy male subjects A Phase I study of the efficacy of cyclosporine has been completed. A total of 42 healthy men were enrolled in the study. The subjects were divided into three groups (subcutaneous, intramuscular and intravenous administration) of 4 subjects. The 50 mM histidine, 125 mM arginine, 0.01% ... Freezing antibody at 60mg / mL in .06% polysorbate 80, 10% sucrose Doses were reconstituted from the dry formulation. For intramuscular and subcutaneous subjects, doses were 1.5 mL each. Blood was collected to determine plasma vedolizumab concentrations, and each set was divided into two injections of 1 mL. The bioavailability of vedolizumab in subjects was determined.

[0318] Serious adverse events or significant infections, clinically significant abnormalities, positive subjective / objective RA No MP checklist or clinically significant ECG findings were reported.

[0319] PK / PD modeling and simulations were completed to determine whether this desired blood Dosage schedules for intravenous and extravascular doses that produce exposures similar to those of intravenous doses to maintain serum concentrations. The painting was decided.

[0320] The absorption profile (Figure 18) showed that intramuscular and subcutaneous dose concentrations generally overlapped. There are no obvious macroscopic differences in the absorption characteristics of these routes of administration. The absolute bioavailability of the drug was approximately 75% and approximately 80% following IM injection. It was.

[0321] Example 14 Modeled subcutaneous administration schedule PK / PD modeling and simulations were completed to assess the efficacy and safety of cyclophosphamide at specific serum concentrations at trough levels. A dosing schedule that produces exposure similar to that of the intravenous dose and an extravascular dose to maintain efficacy was developed. It has been decided.

[0322] The final combined data set (IV, SC and IM data) was CL), median value of distribution (V2), peripheral value of distribution (V3), absorption rate depending on extravascular route The dose constant (KA) and the relative bioavailability of the extravascular dose (F) (compared to intravenous administration) We present a two-compartment linear model parameterized in terms of the allometric effect. CL, V2 and V3 with weight as the only covariate affecting CL and V3 via was included in 3.

[0323] The acceptability and predictability of the models were evaluated using bootstrap parameter estimation, visual prediction checks and The analysis of the model revealed that vedolizumab Body weight was identified as a predictor of PK, and PK variability was attributed to between subjects and between intrasubject components. Ta.

[0324] Once the model is found to be suitable for simulation, the route of administration (IV, IM or S C) to evaluate the effect of dosing frequency (every week, every 2 weeks, every 4 weeks, Simulations were performed to evaluate the effect of a 2-week treatment (once a week, once every 8 weeks, once every 12 weeks, once every 1 ... Based on the relative bioavailability of vedolizumab following IM and SC administration (F = 69.5%) Based on this, doses were selected to achieve similar trough concentrations as the IV dose.

[0325] The simulation will be used to plan doses and schedule medication to harmonize intravenous induction and maintenance regimens. The targets were exposure (area under the serum drug concentration-time curve (AUC)) and toxicity. Both were rough drug concentrations. Tables 40-43 provide the results of the simulations.

[0326] [Table 40]

[0327] [Table 41]

[0328] [Table 42]

[0329] [Table 43]

[0330] Example 15 Phase 2a multiple dose study The Phase 2a multiple dose study will evaluate multiple doses of vedolizumab administered by the subcutaneous route. To evaluate the safety, tolerability, and steady-state PK of vedolizumab compared with an intravenous dosing regimen The relative bioavailability of the dosing regimens can be assessed. Incidence of HAHA after subcutaneous administration To evaluate the effect of neutralizing HAHA and multiple doses of vedolizumab on PD can.

[0331] Patients with ulcerative colitis with a partial Mayo score of 1 to 12 and a CDA greater than 150 Patients with Crohn's disease who have I can be included in the study. Cohorts will be randomized to receive the cohort at weeks 0 and 2. Patients received an induction regimen of vedolizumab (300 mg) administered IV at 100 mg / kg, followed by Any maintenance regimen: Vedolizumab (300 mg) given IV every 4 weeks for weeks 6 to 22; Vedolizumab (300 mg) given IV every 8 weeks for weeks 6 to 22; Vedolizumab (108 mg) administered SC weekly for weeks 6 to 22; Vedolizumab (108 mg) administered SC every 2 weeks from weeks 6 to 22; Receive vedolizumab (165 mg) administered SC every 3 weeks from weeks 6 to 22. can be done.

[0332] Samples were taken pre-dose on day 1, then again on days 1 (12 hours), 2, 3, 5, 8, 15, 2 9, 43, 127, 127 (12 hours), 128, 129, 131, 134, 141, and and 155 C. Patients can be sampled at 155 C and 155 D to assess PK and PD.

[0333] Example 16 Long-term clinical experience with vedolizumab in the treatment of IBD Long-term vedolizumab pharmacokinetics (PK) profile determined after completion of a Phase 2 open-label safety extension study The pharmacodynamics (PD), safety and efficacy of the drug were evaluated. Patients were aged 18-75 years and had ulcerative colitis. Previous participation in a previous PK / PD safety study in patients with colitis or endoscopic and / or histopathological and / or radiological findings within 1 month of screening They had confirmed IBD symptoms for at least 2 months.

[0334] All patients received 2 mg / kg or 6 mg / kg vedolizumab (5 mg / mL antibody, 20 mM citrate / citric acid, 125 mM sodium chloride, 0.05% Polysol Bate 80, pH 6.0 (store at -70°C for long term, -20°C for up to 3 months) and 43 days after the first dose, and then every 8 weeks for a total of 78 weeks. Patients with ulcerative colitis or Crohn's disease who have no history of pulmonary embolism or who have participated in previous clinical trials The patient was suffering from ulcerative colitis.

[0335] Efficacy / Quality of Life (QoL), Partial Mayo Score (PMS), Crohn's Disease Activity Index The study outcomes were assessed using the CDAI and the Inflammatory Bowel Disease Questionnaire (IBDQ). .

[0336] Penalty result Mean pre-infusion vedolizumab concentrations were dose-proportional and remained stable throughout the study was detectable through

[0337] PD results The receptor (%ACT-1+[CD4+CD45R O high] and %MADCAM+[CD4+CD45RO high] were almost completely inhibited.

[0338] Partial Mayo score The mean baseline PMS was significantly higher in treated patients with recurrent ulcerative colitis (2.3). By day 43, PMS was relapsing. The reduction was significant in both relapsing and treatment-naïve ulcerative colitis patients. 15 By day 5, the mean scores for the two groups were similar. Mean PMS declined through day 267. It continued and then it leveled off.

[0339] Crohn's Disease Activity Index The CDAI in CD patients decreased from 294.6 at baseline to 237.7 at day 43. , and continued to decline through day 155 (156.1).

[0340] IBDQ Patients with recurrent ulcerative colitis had the highest mean IBDQ scores at baseline. By day 43, mean IBDQ scores increased in all three disease groups. A continued to increase over time in all three disease groups, and by day 155 in patients with Crohn's disease, In patients with ulcerative colitis who had never had ulcerative colitis and in patients with recurrent ulcerative colitis, the maximum reached.

[0341] C-reactive protein Patients with recurrent ulcerative colitis and Crohn's disease showed a mean C decline up to 155 days Patients with untreated ulcerative colitis showed ulcer had lower mean CRP levels at baseline than patients with recurrent idiopathic colitis (2.28 The mean CRP levels of treatment-naive ulcerative colitis patients were remained relatively constant across all time points.

[0342] Other safety findings No systemic opportunistic infections (including PML) were reported during the study. One patient had a single 72 were JC viremia positive at time point, but all were JCV negative at other time points. Three of the patients (4%) had a positive HAHA outcome (two of these were transiently positive). Studies were conducted to assess the risk of liver toxicity, lymphocytosis or lymphopenia, or other drug-related There was no evidence of changes in clinical laboratory values.

[0343] conclusion Vedolizumab was administered at 2.0 or 6.0 mg / kg once every 8 weeks for up to 78 weeks. , achieving saturation of the target receptors and demonstrating a long-lasting average reduction in disease activity and improved IBD outcomes. Q was generally safe and well tolerated, with acceptable immunogenicity.

[0344] Example 17 Induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis To evaluate the induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis A single study was designed to include two randomized, double-blind, multicenter trials to assess the efficacy and safety of 100 mg / kg / day ... Baseline disease characteristics were comparable across treatment groups.

[0345] Induction tests using intravenous administration were performed using 50 mM histidine, 125 mM arginine, 0 60mg / ml in .06% polysorbate 80, 10% sucrose, pH 6.3 The antibody was reconstituted in a 300 mg dose from a lyophilized formulation of the antibody, and the 6 Vedolizumab was compared with placebo at a weekly endpoint.

[0346] A maintenance study using the same formulation and route as the induction study will compare vedolizumab to placebo However, vedolizumab was administered every 4 weeks, and placebo for vedolizumab was administered every 8 weeks. The endpoint of this study was 52 weeks, and the population of induced responders was analyzed.

[0347] Blood samples were collected to measure vedolizumab concentrations throughout the study. The mean serum concentrations of lizumab were 20-30 μg / mL. The mean trough serum concentrations of vedolizumab at steady state after IV infusion were In the q4wks regimen (4-week regimen), the mean values ​​were between 9 and 13 μg / mL, and in the q4wks regimen (4-week regimen), the mean values ​​were between 3 and 4 μg / mL. The median plasma concentration of vedolizumab at the end of the infusion ranged from 5 to 40 μg / mL. The mean values ​​for the wks regimen were between 98 and 101 μg / mL, whereas the mean values ​​for the q4wks regimen were between 129 and 141 μg / mL. and around 137 μg / mL.

[0348] A summary of the induction and maintenance study responses is provided in Tables 44-47. A significantly larger proportion of patients achieved clinical response, remission, and mucosal healing at 6 weeks compared with placebo. The target of treatment was achieved (Table 44). 39% of the intent-to-treat population in the induction phase had failed prior anti-TNF-α therapy. Clinical response and remission rates were higher in patients with prior anti-TNF failure and in those with Vedolizumab was superior to placebo in both anti-TNF-naïve and anti-TNF-naïve patients. Preliminary analyses of adverse events (AEs), serious AEs, and events leading to study discontinuation over the course of up to two weeks showed The rate of adverse events was significantly higher in the placebo group than in the vedolizumab group. A cohort of patients with a high vedolizumab score achieved clinical remission, mucosal healing, and corticosteroid therapy at 52 weeks. Patients achieved steroid-free remission and durable responses and remissions (Table 45). 32% had failed previous anti-TNF-α therapy. Rates of clinical remission and durable responses vedolizumab was more effective than placebo in both TNF-failed and TNF-naive patients. In the safety population (N=895) from 0 to 52 weeks, the incidence of adverse events (AEs) was The incidence of serious AEs and serious infections was similar between the vedolizumab and placebo groups. No increased rates of clinical or enteric infections were observed in the vedolizumab group.

[0349] [Table 44]

[0350] [Table 45]

[0351] [Table 46]

[0352] [Table 47]

[0353] Example 18 Induction and maintenance of responses and remissions in patients with moderately to severely active Crohn's disease To evaluate the induction and maintenance of response and remission in patients with moderately to severely active Crohn's disease A single study was designed to include two randomized, double-blind, multicenter trials. Baseline disease characteristics were comparable across treatment groups.

[0354] Induction tests using intravenous administration were performed using 50 mM histidine, 125 mM arginine, 0 60mg / ml in .06% polysorbate 80, 10% sucrose, pH 6.3 The antibody was reconstituted in a 300 mg dose from a lyophilized formulation of the antibody, and the 6 Vedolizumab was compared with placebo at a weekly endpoint.

[0355] A maintenance study using the same formulation and route as the induction study will compare vedolizumab to placebo However, vedolizumab was administered every 4 weeks, and placebo for vedolizumab was administered every 8 weeks. The endpoint of this study was 52 weeks, and the population of induced responders was analyzed.

[0356] Surprisingly, the study showed similar results in the Q4 and Q8 groups. A summary of responses from the induction and maintenance studies is provided in Tables 48 to 51. A significantly larger proportion of patients treated with vedolizumab achieved clinical remission and higher response rates The rates of clinical remission and high response were significantly higher in patients with previous TNF failure and in those with Both prior and non-TNF-exposed patients were significantly higher in vedolizumab patients than in placebo. The rates of adverse events (AEs), serious AEs, and serious infections were significantly higher in the vedolizumab and placebo groups. The rates of opportunistic or enteric infections were similar in both groups. No increase in the rate of opportunistic or enteric infections was observed in the vedolizumab group. I couldn't.

[0357] [Table 48]

[0358] [Table 49]

[0359] [Table 50]

[0360] [Table 51]

[0361] Example 19 Induction of responses and remissions in patients with moderately to severely active Crohn's disease A randomized, double-blind, placebo-controlled, multicenter study was completed with 6 weeks (2 trials at 0 and 2 weeks) in patients who had failed TNFα antagonists at week 10 (after 3 doses) and week 10 (after 3 doses) 0 mg dose (lyophilized 50 mM histidine, 125 mM arginine, 0.06% of antibody at 60 mg / ml in polysorbate 80, 10% sucrose, pH 6.3. The study evaluated the induction effect of vedolizumab in 416 patients. Of these, 75% were patients who had failed TNFα antagonists and 25% were patients who had failed TNFα therapy. Demographics and concomitant IBD medications were balanced across treatment groups. Baseline disease characteristics were also consistent across treatment groups, except for baseline disease activity. A balance was maintained over the period.

[0362] The primary endpoint of the study was remission at 6 weeks (%) in the TNF-α antagonist failure population. The key secondary endpoints to be evaluated (sequential study procedure) were 6-week relief of symptoms in the overall population. Response (%) at 10 weeks in the TNF-α antagonist failure group and overall group (H ochberg method), and 6-week and overall populations in the group that failed TNF-α antagonists. Sustained remission (%) at 10 weeks (using Hochberg method) and TNF-α antagonist improved response (%) at 6 weeks in the group that failed IV chemotherapy.

[0363] [Table 52]

[0364] [Table 53]

[0365] [Table 54]

[0366] [Table 55]

[0367] Study shows that patients who fail TNF-α antagonists require three doses to induce remission The remission rate in patients who had failed TNF-α antagonists increased between weeks 6 and 10. The effect was statistically significant, but only in the vedolizumab group (not in the placebo group). Remission rates for drug-naive patients did not increase substantially between weeks 6 and 10. Among patients with high disease severity who failed TNF-α antagonists, 43% had TNFR. They never responded to Fα antagonists, and 45% lost response.

[0368] Example 20: Stability Stability of different anti-α4β7 antibody formulations over a period of 6 to 24 months at 5°C The results were examined (Tables 6 and 7). The formulations with a pH of 6.0 to 6.2 showed a significant improvement after 6 months and 24 months. After 1 month, approximately less than 4% of the major species was degraded.

[0369] The stability of different anti-α4β7 antibody formulations was examined by SEC for up to 24 months. (Tables 4 and 5). The formulation had a 0.1-0.2% change in aggregates after 2 years, but the protein concentration at 160 mg / mL was The formulation containing 25 mM citrate and 25 mM glycerol resulted in approximately 0.3% aggregates after 2 years. The citrate-free 60, 110 or 160 mg / mL protein There was a 0.6-1.1% increase in aggregates for formulations containing citric acid. However, for the formulations not containing histidine examined at 12 and 24 months, the difference was approximately 0. There was an increase in aggregates ranging from 3 to 0.4%.

[0370] Example 21: To determine the effect of vedolizumab on the CD4:CD8 ratio Reconstituted from a lyophilized formulation in 10% sucrose and diluted in 0.9% saline infusion system Healthy subjects aged 18-45 years were treated with a single dose of 450 mg vedolizumab. Cerebrospinal fluid (CSF) was measured by lumbar puncture before (baseline) and 5 weeks after a single dose of vedolizumab. CSF) was collected. Each subject served as their own control.

[0371] At the 5-week time point, MS patients treated with natalizumab had CSF demonstrated an effect on the CD4+:CD8+ lymphocyte ratio and a reduction in the number of brain lesions Previous studies have shown that Neurol. 2006;59:743-747. Miller et al. N Engl J Med. 2003;348(1):15-23) and also at week 5, a 450 mg dose of vedolizumab was sufficient to saturate the target. Exceeds estimated steady-state trough levels associated with a Phase 3 dose study of 300 mg every 4 weeks The 5 week time point was chosen because it provides serum levels that can be used to

[0372] Approximately 15 mL of CSF was obtained from each subject for immunophenotyping. ≤10 RBCs / μL per sample (to minimize peripheral blood contamination); negative CSF culture results; adequate T lymphocyte counts in each flow cytometry sample; and CSF samples were included in the analysis if they met the criteria of no detectable serum antibodies to izumab.

[0373] Median (34.80 μg / mL) and individual serum vedolizumab concentrations at week 5 (2 4.9-47.9 μg / mL) is the projected steady-state dose for Phase 3 dosing regimens. The trough concentration was higher than that of the normal state (approximately 24 μg / mL). Receptor saturation was observed at 5 weeks as measured by MAdCAM-1-Fc, and end points This indicated saturation of the target with vedolizumab at the time of evaluation.

[0374] Vedolizumab was not detected in any CSF samples (detection limit = 0.125 μg). g / mL).

[0375] Effects on numbers and ratios of CD4+ and CD8+ lymphocytes Vedolizumab did not significantly reduce the CD4+:CD8+ ratio (Table 56). None had a post-dose CD4+:CD8+ ratio of <1 (p<0.0001, one-tailed t-test). Vedolizumab increased CSF CD4 + or significantly reduce the number of CD8+ T lymphocytes In addition, there were no significant changes in the percentage of CD4+ or CD8+ T lymphocytes in the CSF. There were no significant changes in peripheral blood WBC, CD4+ and CD8+ memory T lymphocytes (Table 57). No significant changes were observed (Table 58).

[0376] [Table 56]

[0377] [Table 57]

[0378] [Table 58]

[0379] summary Vedolizumab increased CSF CD4+ and There was no effect on CD8+ cell counts or the CD4+:CD8+ ratio. There was no decline in the CSF CD4+:CD8+ ratio below 1 after treatment. In addition, CD4+ was not detected in total WBCs or memory T lymphocytes in peripheral blood. There were no changes observed in the CD8+ and CD8+ subsets. Saturation occurred in all subjects at the end point assessment. CSF CD4+:CD8+ lymphocytes The levels and ratios were similar to those previously reported in the literature.

[0380] These results suggest that IL-16 may be a potent inhibitor of CNS immune response in monkeys, both in physiological CNS immune surveillance and in pathological CNS inflammation. This is consistent with vedolizumab's lack of efficacy in treating bronchitis.

[0381] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, the accompanying drawings, in which: Various changes in form and detail may be made without departing from the scope of the invention encompassed by the present invention. It will be understood by those skilled in the art that may be done therein.

[0382] [Table 59-1] [Table 59-2]

[0383] Aspects of the present invention include the following. [1] A stable liquid pharmaceutical formulation comprising an anti-α4β7 antibody, an antioxidant or a chelating agent, and at least one A stable liquid pharmaceutical formulation comprising a mixture of at least one free amino acid, the formulation being in liquid form. Drug preparations. [2] The molar ratio of the anti-α4β7 antibody to the antioxidant or chelating agent is about 1:4 to about 1:100. A stable liquid pharmaceutical formulation according to [1]. [3] After 12 months at 5° C., the stable liquid formulation has less than about 1.0% aggregate formation. [1] A stable liquid pharmaceutical formulation according to the present invention. [4] The stable liquid pharmaceutical preparation according to [3], wherein the formulation has less than about 0.2% aggregate formation. Agent. [5] The stable liquid pharmaceutical composition according to [1], wherein the antioxidant or chelating agent is a citrate salt. Agent. [6] The stable liquid pharmaceutical formulation according to [1], wherein the chelating agent is EDTA. [7] The free amino acid is histidine, alanine, arginine, glycine, glutamine The stable liquid pharmaceutical composition according to [1], Agent. [8] The stable liquid pharmaceutical formulation according to [1], wherein the formulation further comprises a surfactant. [9] The molar ratio of the antioxidant or chelating agent to the surfactant is about 3:1 to about 156:1. 8] A stable liquid pharmaceutical formulation according to the present invention.

[10] The surfactant is polysorbate 20, polysorbate 80, poloxamer, and The stable liquid pharmaceutical formulation according to [8], wherein the pharmaceutical composition is selected from the group consisting of:

[11] The stable liquid pharmaceutical preparation according to [1], wherein the preparation has a pH between about 6.1 and about 7.0. Drug preparations.

[12] The stable liquid pharmaceutical preparation according to [1], wherein the preparation has a pH of about 6.5 to about 6.8. Drug preparations.

[13] The formulation comprises at least about 60 mg / ml to about 160 mg / ml of an anti-α4β7 antibody. The stable liquid pharmaceutical formulation according to [1], comprising the body.

[14] The method according to [1], wherein the formulation contains at least about 160 mg / ml of an anti-α4β7 antibody. The stable liquid pharmaceutical formulation described above.

[15] A stable liquid pharmaceutical formulation having a concentration of at least about 60 mg / ml to about 160 mg / ml. 1 of an anti-α4β7 antibody, a buffering agent, and at least about 5 mM citrate, said formulation comprising A stable liquid pharmaceutical formulation which is a liquid formulation.

[16] The stable liquid pharmaceutical formulation according to

[15] , wherein the buffering agent is a histidine buffer.

[17] At least about 160 mg / ml of anti-α4β7 antibody and at least about 5 mM of quencher A stable liquid pharmaceutical formulation comprising a phosphate salt.

[18] The stable liquid pharmaceutical preparation according to

[17] , wherein the preparation further contains polysorbate 80. Agent.

[19] A stable liquid pharmaceutical formulation comprising an anti-α4β7 antibody, citrate, histidine, and aryl. A stable liquid pharmaceutical preparation comprising a mixture of ginseng and polysorbate 80, the preparation being in liquid form. Agent.

[20] The formulation is selected from the group consisting of a vial, a cartridge, a syringe, and an automatic injector. The stable liquid pharmaceutical formulation according to

[19] , present in a selected container.

[21] A stable liquid formulation selected from any one of the formulations in Table 2.

[22] The stable antibody according to any one of [1] to

[21] , wherein the antibody is vedolizumab. Liquid pharmaceutical formulations.

[23] Any one of [1] to

[21] , wherein the formulation is for subcutaneous administration or intramuscular administration. Item 1. A stable liquid pharmaceutical formulation according to item 1.

[24] A method for treating inflammatory bowel disease, comprising administering to a patient in need thereof the composition described in [1]. Administering a pharmaceutical formulation of

[25] The method according to

[24] , wherein the administration is subcutaneous administration.

[26] The method according to

[24] , wherein the administration is self-administration.

[27] A manufactured article comprising a container, a stable liquid according to any one of [1] to

[21] , An article of manufacture that includes a pharmaceutical formulation and instructions for its use.

[28] The article of manufacture of

[27] , wherein the article is a pre-filled syringe.

[29] A method of treating a human patient suffering from inflammatory bowel disease, the method comprising: Humanized α4β7 integrin-binding specificity in human patients suffering from inflammatory bowel disease administering an immunoglobulin or an antigen-binding fragment thereof, The following dosing schedule: (a) 165 mg of humanized immune globulin as a subcutaneous injection once every two days for six doses. a first administration of the agonist or an antigen-binding fragment thereof; (b) followed by 165 mg subcutaneously every 2 weeks or every 4 weeks as needed The seventh and subsequent administrations of the humanized immunoglobulin or antigen-binding fragment thereof are administering to the patient a globulin or an antigen-binding fragment thereof; the dosing regimen induces a clinical response and clinical remission of the inflammatory bowel disease in the patient; Furthermore, humanized immunoglobulins or antigen-binding fragments comprise an antigen-binding region of non-human origin and a human and wherein the humanized immunoglobulin or antigen-binding fragment comprises at least a portion of an antibody of human origin, and an antigen-binding region having binding specificity for the β7 complex, the antigen-binding region comprising: 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 .

[30] Patients may receive at least one of the following: immunomodulators, tumor necrosis factor-α antagonists, or a combination of these. Lack of adequate response to, loss of response to, or inability to tolerate treatment with at least one The method described in this study was not tolerated in any clinical trials

[29] .

[31] The method according to

[29] , wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[32] The method according to

[31] , wherein the inflammatory bowel disease is ulcerative colitis.

[33] Inflammatory bowel disease is defined as moderately to severely active ulcerative colitis.

[31] Method of posting.

[34] The treatment regimen was shown to promote mucosal healing in patients suffering from moderate to severe active ulcerative colitis. The method according to claim 33,

[35] The dosing regimen reduces, eliminates, or reduces and eliminates the use of corticosteroids by the patient. The method according to claim 29, wherein

[36] Patients had previously received at least one corticosteroid for inflammatory bowel disease. The method described in

[29] was used in patients who had received treatment with

[37] A humanized immunoglobulin or an antigen-binding fragment thereof is administered at a concentration of about 1.0 mg / mL to about 1.4 mg / mL. The method according to claim 29, wherein the compound is administered in a final dosage form at a concentration between 100 mg / mL and 200 mg / mL.

[38] The humanized immunoglobulin or antigen-binding fragment thereof is administered at a final dose of about 1.2 mg / mL. The method according to claim 37, wherein the compound is administered in a dosed form.

[39] The dosing regimen altered the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving the treatment.

[29] A method according to the present invention.

[40] A dosing regimen for treating inflammatory bowel disease, comprising: The method comprises administering to a patient suffering from inflammatory bowel disease a compound that binds specifically to human α4β7 integrin. administering to the patient a humanized immunoglobulin or antigen-binding fragment thereof having the activity of A humanized immunoglobulin or antigen-binding fragment is an antibody that has an antigen-binding region of non-human origin and an antibody of human origin. The humanized immunoglobulin or antigen-binding fragment thereof comprises at least a portion of The drug is administered to patients according to the protocol. The patient was administered a medicament with binding specificity for human α4β7 integrin for patients suffering from inflammatory bowel disease. The method includes administering a humanized immunoglobulin or antigen-binding fragment thereof to a mammalian animal, The immunoglobulin or antigen-binding fragment thereof may be administered in the following dosage regimen: (a) 165 mg of humanized immune globulin as a subcutaneous injection once every two days for six doses. a first administration of the agonist or an antigen-binding fragment thereof; (b) followed by 165 mg subcutaneously every 2 weeks or every 4 weeks as needed the seventh and subsequent administrations of the immunized immunoglobulin or antigen-binding fragment thereof; is administered to the patient according to the dosing regimen induces a clinical response and clinical remission of the inflammatory bowel disease in the patient; Furthermore, humanized immunoglobulins or antigen-binding fragments comprise an antigen-binding region of non-human origin and a human and wherein the humanized immunoglobulin or antigen-binding fragment comprises at least a portion of an antibody of human origin, and an antigen-binding region having binding specificity for the β7 complex, the antigen-binding region comprising: 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 plan.

[41] Patients may receive at least one of the following: immunomodulators, tumor necrosis factor-α antagonists, or a combination of these. Lack of adequate response to, loss of response to, or inability to tolerate treatment with at least one The dosing regimen described in

[40] was not tolerated.

[42] The method according to

[40] , wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Picture.

[43] The administration regimen described in

[41] , wherein the inflammatory bowel disease is ulcerative colitis.

[44] As described in

[43] , inflammatory bowel disease is moderately to severely active ulcerative colitis. The dosing regimen is as follows.

[45] The treatment regimen was shown to promote mucosal healing in patients suffering from moderate to severe active ulcerative colitis. The dosing regimen described in

[44] produces

[46] The dosing regimen reduces, eliminates, or reduces and eliminates the use of corticosteroids by the patient. The dosing regimen described in

[40] results in elimination of

[47] Patients had previously received at least one corticosteroid for inflammatory bowel disease. The dosing regimen was as described in

[40] .

[48] ​​A humanized immunoglobulin or an antigen-binding fragment thereof is administered at a concentration of about 1.0 mg / mL to about 1.4 mg / mL. The dosing regimen described in

[40] is administered in a final dosage form at a concentration between 100 mg / mL.

[49] The humanized immunoglobulin or antigen-binding fragment thereof is administered at a final dose of about 1.2 mg / mL. The dosage regimen described in

[48] is administered in a given form.

[50] The dosing regimen alters the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving the treatment. The dosing regimen described in

[40] cannot be used.

[51] The patient is 65 years of age or older, and the patient does not require adjustment of the dosing regimen. The method described in

[40] .

[52] The patient is 65 years of age or older, and the patient does not require adjustment of the dosing regimen.

[40] The dosing regimen described.

[53] A method of treating a human patient suffering from inflammatory bowel disease, the method comprising administering to a patient a human α4β Humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for integrin 7 to a patient suffering from inflammatory bowel disease, The humanized immunoglobulin or antigen-binding fragment thereof may be administered in the following dosage regimen: (a) about 20 to about 30 μg / mL of humanized immunoglobulin within about 6 weeks of the first administration, or Sufficient humanized immunoglobulin or an antigen-binding fragment thereof to achieve a mean trough serum concentration multiple induction phase doses of the antigen-binding fragment; (b) then administering a humanized immunoglobulin at about 9 to about 13 μg / mL or about 35 to 40 μg / mL; Required to maintain the mean steady-state trough serum concentration of Brin or an antigen-binding fragment thereof Multiple maintenance phase doses of humanized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the dosing regimen induces a clinical response and clinical remission of the inflammatory bowel disease in the patient; Furthermore, humanized immunoglobulins or antigen-binding fragments comprise an antigen-binding region of non-human origin and a human and wherein the humanized immunoglobulin or antigen-binding fragment comprises at least a portion of an antibody of human origin, and an antigen-binding region having binding specificity for the β7 complex, the antigen-binding region comprising: 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 .

[54] The method of

[53] , wherein the dose is administered subcutaneously or intramuscularly.

[55] The induction dose is administered intravenously, and the maintenance dose is administered subcutaneously or intramuscularly. The method described in

[53] .

[56] The induction phase doses were once every 2 days, approximately once every week, and approximately once every 2 weeks. The method according to claim 54, wherein the administration is performed at intervals selected from the group consisting of:

[57] The maintenance dose is approximately once every week, approximately once every 2 weeks, or approximately once every 3 weeks. once every 4 weeks, once every 6 weeks, once every 8 weeks, and once every 10 weeks

[54] The method according to claim 54, wherein the compound is administered at intervals selected from the group consisting of once every 10 days and once every 20 days.

[58] Induction doses were administered at approximately weeks 0 and 2, and maintenance doses were administered at approximately week 6. Started, approximately once every week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks once every 6 weeks, once every 8 weeks, and once every 10 weeks. The method according to claim 55, wherein the dose is administered at an interval selected from the group consisting of:

[59] The method according to

[54] , wherein the dose is self-administered.

[60] The method according to

[55] , wherein the maintenance phase doses are self-administered.

[61] The patient according to

[54] , each dose of which contains approximately 165 mg of humanized immunoglobulin. Law.

[62] Each maintenance dose contains approximately 165 mg of humanized immunoglobulin.

[55] The method described.

[63] Patients may receive at least one of the following: immunomodulators, tumor necrosis factor-α antagonists, or a combination of these. Lack of adequate response to, loss of response to, or inability to tolerate treatment with at least one The method was not tolerated in any clinical trials

[53] .

Claims

1. 1. A pharmaceutical composition for treating inflammatory bowel disease in a human patient, the composition comprising: Complementarity determining region 1 (CDR1) shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 9, and and a heavy chain variable region comprising the CDR3 set forth in SEQ ID NO: 10, and a heavy chain variable region comprising the CDR3 set forth in SEQ ID NO:

11. CDR1, CDR2 shown in SEQ ID NO:12 and CDR3 shown in SEQ ID NO:13 The antibody comprises a humanized anti-α4β7 antibody comprising a light chain variable region, the antibody being administered to a human patient every two weeks. A pharmaceutical composition to be administered subcutaneously at a dose of 108 mg.

2. 1. A pharmaceutical composition for treating inflammatory bowel disease in a human patient, the composition comprising: Complementarity determining region 1 (CDR1) shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 9, and and a heavy chain variable region comprising the CDR3 set forth in SEQ ID NO: 10, and a heavy chain variable region comprising the CDR3 set forth in SEQ ID NO:

11. CDR1, CDR2 shown in SEQ ID NO:12 and CDR3 shown in SEQ ID NO:13 and a humanized anti-α4β7 antibody comprising a light chain variable region, the antibody being administered at a dose of 300 mg in a clinical setting. Human patients are administered intravenously until a response is achieved, followed by subcutaneous administration of 108 mg of the antibody. A pharmaceutical composition in which a dose is administered to a human patient such that a clinical response is maintained.

3. Clinical responses were evaluated following intravenous administration of a first 300 mg dose of the antibody and 2 weeks after administration of the first dose.

3. The pharmaceutical composition of claim 2, followed by administration of a second 300 mg dose of said antibody. 。

4. A clinical response was observed following administration of a third 300 mg dose of the antibody four weeks after the second dose. The pharmaceutical composition according to claim 3, wherein

5. A 108 mg subcutaneous dose of the antibody is initiated at least 14 weeks after the first dose. The pharmaceutical composition according to claim 4.

6. 6. The method of claim 2, wherein a subcutaneous dose of 108 mg of the antibody is administered every two weeks. The pharmaceutical composition described above.

7. 1. A pharmaceutical composition for treating inflammatory bowel disease in a human patient, the composition comprising: CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO:9 and CDR3 shown in SEQ ID NO:10 and a heavy chain variable region comprising CDR3 as set forth in SEQ ID NO:11, CDR1 as set forth in SEQ ID NO:1 and a light chain variable region comprising CDR2 represented by SEQ ID NO: 13 and CDR3 represented by SEQ ID NO:

14. the antibody comprising a first intravenous dose of 300 mg of the antibody, a second dose of 300 mg of the antibody administered intravenously two weeks after the first dose, and and a 108 mg dose of the antibody administered subcutaneously 6 weeks after the dose and every 2 weeks thereafter. A pharmaceutical composition to be administered according to a dosing regimen.

8. The antibody comprises a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO:2 and an amino acid sequence comprising SEQ ID NO:

4. The pharmaceutical composition according to any one of claims 1 to 7, comprising a light chain variable region comprising amino acids 20 to 131. 。

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody is of the IgG1 isotype.

10. The antibody comprises a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:2 and a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:

4.

8. The pharmaceutical composition of claim 1, comprising a light chain comprising 0 to 238.

11. The pharmaceutical composition of any one of claims 1 to 7, wherein the antibody is vedolizumab.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is prepared for self-administration. The pharmaceutical composition described above.

13. The pharmaceutical composition is present in a vial, cartridge, syringe or automatic injector. The pharmaceutical composition according to any one of claims 1 to 12,

14. The method according to any one of claims 1 to 13, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. A pharmaceutical composition comprising:

15. The pharmaceutical composition according to claim 14, wherein the ulcerative colitis is moderately to severely active ulcerative colitis. composition.

16. the dosing regimen results in mucosal healing in human patients with ulcerative colitis; A pharmaceutical composition according to claim 14 or 15.

17. A human patient is receiving treatment with at least one corticosteroid for inflammatory bowel disease. The pharmaceutical composition according to any one of claims 1 to 16, wherein the patient has previously received

18. The patient is receiving at least one of an immunomodulatory agent, a corticosteroid, or a tumor necrosis factor-α antagonist. have lacked an adequate response to treatment with one of the The pharmaceutical composition according to any one of claims 1 to 16, for which 。

19. The dosing regimen results in the reduction, elimination, or reduction and elimination of corticosteroid use. The pharmaceutical composition according to any one of claims 1 to 16,

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