Formulations for anti-α4β7 antibodies

Abstract

To provide a formulation for anti-α4β7 antibodies. [Solution] To maintain clinical remission of ulcerative colitis or Crohn's disease in human patients A composition having binding specificity to human α4β7 integrin. The composition contains a dose of 300 mg of the antibody, and is administered to human patients every 4 weeks or every 8 weeks. The antibody is administered to the complementarity-determining region 1 (CDR1) shown in SEQ ID NO: 8, and to SEQ ID NO: 9. It includes a heavy chain variable region including CDR2 shown and CDR3 shown in Sequence ID No. 10, and Sequence ID No. Includes CDR1 shown in 11, CDR2 shown in sequence number 12, and CDR3 shown in sequence number 13. A composition containing a light chain variable region.

Description

Technical Field

[0001] The present invention relates to a formulation for an anti-α4β7 antibody.

[0002] Related Applications This application claims the benefit of U.S. Provisional Patent Application 61 / 585,859, filed on January 12, 2012, U.S. Provisional Patent Application 61 / 550,545, filed on October 24, 2011, and U.S. Provisional Patent Application 61 / 481,533, filed on May 2, 2011. The entire contents of the foregoing applications are incorporated herein by reference.

[0003] Sequence Listing This application contains a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on April 30, 2012, is named 92596615.txt and is 17,024 bytes in size.

Background Art

[0004] Advances in biotechnology have made it possible to produce various proteins for pharmaceutical applications using recombinant DNA. Proteins are much larger and more complex than conventional inorganic and organic drugs (i.e., having a complex three-dimensional structure in addition to multiple functional groups), and formulating such proteins presents special problems. To maintain the biological activity of a protein, the formulation must preserve the structural integrity of at least the core sequence of the protein's amino acids while simultaneously protecting the multiple functional groups of the protein from degradation. Proteins can be plagued by a lack of stability, and monoclonal antibodies and polycl onal antibodies can be particularly unstable.

[0005] Ronar's antibodies can be particularly relatively unstable (see, e.g., Wang, et al., Non-Patent Document 1). A number of formulation options are available, and one approach or system is not suitable for all proteins. Several factors to be considered have been reported (see, e.g., Wang et al.).

[0005] A number of features can affect the stability of proteins. In fact, even in the case of purified antibodies, the antibody structure can be heterogeneous, which further complicates the formulation of such systems. Furthermore, the excipients included in antibody formulations preferably minimize potential immune responses as much as possible.

[0006] In the case of antibodies, preservation of structural integrity is even more important. In the protein degradation pathway there can be involvement of chemical instability (i.e., processes where protein modification by bond formation or cleavage resulting in new chemical entities is involved) or physical instability (i.e., changes in the higher-order structure of the protein). Chemical instability appears, for example, in deamidation, isomerization, hydrolysis, oxidation, fragmentation, glycan β-elimination or disulfide conversion. Physical instability results, for example, from denaturation, aggregation, precipitation or absorption. The four most common protein degradation pathways are protein fragmentation, aggregation, deamidation and oxidation. The consequences of chemical or physical instability of therapeutic proteins include a decrease in the effective dose, for example, a decrease in the safety of the treatment due to stimulation or immunoreactivity, and frequent manufacturing due to a short shelf life. .

[0007] Freeze-drying is a commonly employed technique for preserving proteins; freeze-drying It helps to remove moisture from the protein preparation. Freeze-drying or freeze-drying is a drying process. The substance to be processed is first frozen, and then the ice or frozen solvent is removed by sublimation under vacuum. This is the process of drying. Excipients are added to the formulation before freeze-drying, and the protein is dried during the freeze-drying process. It can stabilize and / or improve the stability of lyophilized protein preparations (non Patent literature 2 and non-patent literature 3).

[0008] Several publications generally disclose various methods for treating inflammatory bowel disease, We provide a medication scheme for administering drugs designed to treat colorectal diseases. For example, Patent Document 1 describes how leukocytes express mucosal vascular adrenergicsin and MAdCAM. The report discloses the treatment of diseases related to the mobilization of leukocytes into the gastrointestinal tract as a result of the binding of these substances. Patent Document 2 describes a method for treating diseases related to leukocyte infiltration in mucosal tissue and α4β7 inhibitors. Human or humanized immunoglobulins or antigen binding with binding specificity to glycin. It discloses the administration of an effective amount of the fragment to humans. Patent Document 2 further discloses various doses (for example) For example, 0.15, approximately 0.5, approximately 1.0, approximately 1.5, or approximately 2.0 mg of immunoglobulin per kg of body weight. The text describes the robulin (or fragments) and various administration intervals (7, 14, 21, 28, and 30 days). However, the aforementioned patents and publications do not pertain to specific formulations of anti-α4β7 antibodies or the present specification. The document does not disclose the specific dosage and administration plan to be claimed. Importantly, The aforementioned patent is the treatment method described and claimed herein (supported by clinical trial data). The formulation, dosage, and administration plan provided are not disclosed. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] WO96 / 24673 [Patent Document 2] US2005 / 0095238 [Non-patent literature]

[0010] [Non-Patent Document 1] J. Pharm Sci. 96:1-26 (2007) [Non-Patent Document 2] Pikal M., Biopharm. 3(9)26-30 (1990) [Non-Patent Document 3] Arakawa et al. Pharm. Res. 8(3):285-291 (1991) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The antibody preparation of the present invention inhibits leukocytes that bind to cells expressing MAdCAM. Therefore, these may be useful in treating inflammatory bowel disease in patients. To discover suitable dosages and administration plans for compounds, and to maintain them in a stable and convenient form over a long period of time. A preparation that produces stable, therapeutically effective blood levels of antibody preparations, preferably an intravenous preparation. There is an urgent need. [Means for solving the problem]

[0012] Due to its instability, the present invention is sensitive to deamidation, oxidation, isomerization and / or aggregation. Non-reducing sugars and at least one A as excipients useful for formulating anti-α4β7 preparations Regarding the identification of amino acids. The formulation improves stability, reduces aggregate formation, and contains antibodies. It slows down the decomposition.

[0013] Therefore, in the first aspect, the present invention relates to a non-reducing sugar, an anti-α4β7 antibody, and at least one asexual This relates to a stable formulation containing a mixture of amino acids, a non-reducing sugar, and an anti-α4β7 antibody. The molar ratio (moles:moles) exceeds 600:1. The formulation is either a liquid formulation or a dry formulation (for example). It may be (freeze-dried). The formulation may also contain a buffering agent. Some implementations In terms of form, non-reducing sugars include mannitol, sorbitol, sucrose, trehalose, or It's a combination of those.

[0014] In some embodiments, the free amino acids of the formulation are histidine, alanine, arginine, and glycerin. Lysine, glutamic acid, or a combination thereof. The formulation is approximately 50 mM to 175 mM. The formulation may contain free amino acids between M. The formulation is between approximately 100 mM and approximately 175 mM. It can contain free amino acids. The molar ratio of free amino acids to antibody is small. It can also be 250:1.

[0015] The formulation may also contain a surfactant. The surfactant is polysorbate 20. It may be polysorbate 80, polyxamer, or a combination thereof.

[0016] In some embodiments, the formulation can minimize the immunogenicity of the anti-α4β7 antibody.

[0017] For example, a dry formulation should be kept at 40°C and 75% relative humidity (RH) for at least 3 It can remain stable for months.

[0018] In another embodiment, the formulation is freeze-dried, and before freeze-drying, at least about 5% to about 10% anti-α Contains 4β7 antibody. The formulation should contain at least approximately 6% anti-α4β7 antibody before lyophilization. This is possible. A formulation can be reconstituted from a lyophilized formulation (for example, a stable liquid). (It can be reconstituted to include a body formulation.)

[0019] In another embodiment, the present invention relates to a non-reducing sugar, an anti-α4β7 antibody, and at least one free amino acid This relates to a stable formulation containing a mixture of acids, and the molar ratio of non-reducing sugar to anti-α4β7 antibody ( The molar ratio (mol:mol) exceeds 600:1, and the molar ratio of free amino acids to anti-α4β7 antibody (mol:mol) The ratio of (L) exceeds 250:1.

[0020] In another embodiment, the present invention relates to a non-reducing sugar, an anti-α4β7 antibody, and at least one free amino acid This relates to a stable liquid formulation containing an aqueous solution with acid, and involves a non-reducing sugar and an anti-α4β7 antibody. The molar ratio (moles:moles) exceeds 600:1. Furthermore, in a further embodiment, the present invention At least approximately 40 mg / ml to approximately 80 mg / ml of anti-α4β7 antibody, and at least approximately 50 ~175 mM of 1 or more amino acids, and at least approximately 6% to at least approximately 10% (w / v) This relates to a liquid formulation containing sugar. The liquid formulation may also contain a buffering agent. In some embodiments, The liquid formulation also includes a metal chelating agent. In some embodiments, the liquid formulation also includes an antioxidant.

[0021] In another embodiment, the present invention provides at least about 60 mg / ml of anti-α4β7 antibody and at least It also contains approximately 10% (w / v) non-reducing sugars and at least 1 or more amino acids at a concentration of approximately 125 mM. Regarding liquid formulations.

[0022] In another embodiment, the present invention provides at least about 60 mg / ml of anti-α4β7 antibody and at least It also contains approximately 10% (w / v) non-reducing sugars and at least 1 or more amino acids at a concentration of approximately 175 mM. Regarding liquid formulations.

[0023] Furthermore, in a further embodiment, the present invention relates to a non-reducing sugar, an anti-α4β7 antibody, and histidine. , a dry, for example, freeze-dried mixture containing arginine and polysorbate 80 This also relates to the formulation, which is in solid form, and the molar ratio of non-reducing sugar to anti-α4β7 antibody. The ratio (moles:moles) exceeds 600:1.

[0024] Furthermore, in a further embodiment, the present invention relates to a non-reducing sugar, an anti-α4β7 antibody, and histidine. The present invention relates to a liquid formulation comprising a mixture of arginine and polysorbate 80. In this embodiment, The molar ratio (moles:moles) of non-reducing sugars to anti-α4β7 antibodies exceeds 600:1. Furthermore, the formulation The molar ratio (moles:moles) of arginine to anti-α4β7 antibody in this study exceeds 250:1.

[0025] In another embodiment, the present invention maintains the product temperature below the collapse temperature during primary drying. The present invention relates to a method for preparing the formulations described herein, including the annealing step. It can also be included.

[0026] In one embodiment, the present invention relates to a method for treating human patients suffering from inflammatory bowel disease. Therefore, this method involves administering human α4β7 integrin to human patients suffering from inflammatory bowel disease. The process involves administering a humanized immunoglobulin or its antigen-binding fragment having binding specificity to the target. Including, humanized immunoglobulins or antigen-binding fragments, the antigen-binding region is of non-human origin and human origin The source antibody contains at least a portion of the humanized immunoglobulin or its antigen-binding fragment, and the following Administration plan: (a) 300 mg of humanized immunoglobulin or its antigen as intravenous infusion (b) Initial administration of the binding fragment, and (b) subsequent administration as intravenous infusion approximately two weeks after the initial administration. (c) a second subsequent administration of 00 mg of humanized immunoglobulin or its antigen-binding fragment, then Approximately 6 weeks after the initial dose, 300 mg of humanized immunoglobulin was administered intravenously. (d) the third subsequent administration of the antigen-binding fragment, and the third subsequent administration of the humanized antibody. 300 mg of humanized immunoglobulin as an intravenous infusion every 4 or 8 weeks, if necessary. The patient is administered brin or its antigen-binding fragment in accordance with a fourth subsequent dose, and the administration plan is performed according to the patient's It induces clinical responses and clinical remission in individuals with inflammatory bowel disease; and further, humanized The immunoglobulin or antigen-binding fragment has binding specificity to the α4β7 complex; antigen binding The region is one of the three complementarity-determining regions of the light chain variable region of the amino acid sequence shown below (CDR1, C DR2 and CDR3) and the three complementarity determination regions of the heavy chain variable region (CDR1, CDR2 and (CDR3): Light chain CDR1: Sequence number 9, CDR2: Sequence number 10, CDR3: Sequence number No. 11; Heavy chain: CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: Includes 14.

[0027] In another embodiment, the present invention relates to a dosage regimen for the therapeutic use of inflammatory bowel disease. Yes, and this administration plan involves administering human α4β7 integrin to human patients suffering from inflammatory bowel disease. The process involves administering a humanized immunoglobulin or its antigen-binding fragment having binding specificity to the target. Including, humanized immunoglobulins or antigen-binding fragments, the antigen-binding region is of non-human origin and human origin The source antibody contains at least a portion of the humanized immunoglobulin or its antigen-binding fragment, and the following Administration plan: (a) 300 mg of humanized immunoglobulin or its antigen as intravenous infusion (b) Initial administration of the binding fragment, and (b) subsequent administration as intravenous infusion approximately two weeks after the initial administration. (c) a second subsequent administration of 00 mg of humanized immunoglobulin or its antigen-binding fragment, then Approximately 6 weeks after the initial dose, 300 mg of humanized immunoglobulin was administered intravenously. (d) the third subsequent administration of the antigen-binding fragment, and the third subsequent administration of the humanized antibody. 300 mg of humanized immunoglobulin as an intravenous infusion every 4 or 8 weeks, if necessary. The patient is administered brin or its antigen-binding fragment in accordance with a fourth subsequent dose, and the administration plan is performed according to the patient's It induces clinical responses and clinical remission in individuals with inflammatory bowel disease; and further, humanized The immunoglobulin or antigen-binding fragment has binding specificity to the α4β7 complex; antigen binding The region is one of the three complementarity-determining regions of the light chain variable region of the amino acid sequence shown below (CDR1, C DR2 and CDR3) and the three complementarity determination regions of the heavy chain variable region (CDR1, CDR2 and (CDR3): Light chain: CDR1: Sequence ID 9, CDR2: Sequence ID 10, CDR3: Sequence Number 11; Heavy chain: CDR1: Sequence ID 12, CDR2: Sequence ID 13, CDR3: Sequence ID Includes No. 14.

[0028] In some embodiments, the treatment method, dosage, or administration plan with an anti-α4β7 antibody preparation is anti-α4β The immunogenicity of the 7 antibodies can be suppressed as much as possible.

[0029] Patients may be taking immunomodulators, tumor necrosis factor (TNF-α) antagonists, or combinations thereof. At the very least, there was a lack of appropriate response to treatment, or a lack of response to it. Or, it's possible that they were not accepting of it.

[0030] Inflammatory bowel disease can be Crohn's disease or ulcerative colitis. Inflammatory bowel disease is moderate. It can range from mild to severe active ulcerative colitis.

[0031] The treatment plan is for patients suffering from moderate to severe active ulcerative colitis, ultimately resulting in mucosal healing. It can bring about healing.

[0032] The patient had previously been treated with at least one corticosteroid for inflammatory bowel disease. It is possible that this was the case. The treatment plan involves reducing and eliminating the patient's use of corticosteroids. Alternatively, reduction and elimination may result.

[0033] In some embodiments, the humanized immunoglobulin or its antigen-binding fragment is approximately 1.0 mg / mL Humanized immunoglobulin is administered in its final dose form at a concentration between approximately 1.4 mg / mL. Alternatively, the antigen-binding fragment can be administered in a final dose form of approximately 1.2 mg / mL. Humanized immunoglobulin or antigen-binding fragments can be administered to the patient in approximately 30 minutes.

[0034] Humanized immunoglobulin or its antigen-binding fragments are reconstituted from lyophilized preparations. It is possible.

[0035] Humanized immunoglobulin or its antigen-binding fragments are reconstituted to form a stable liquid formulation. It is possible.

[0036] In some embodiments, the administration plan involves CD4 in the cerebrospinal fluid of the patient receiving the treatment and The ratio of CD8 will not be changed.

[0037] Patients can be 65 years of age or older and do not require adjustments to the administration plan.

[0038] In other words, the gist of this invention is, [1] A set of treatments for maintaining clinical remission of ulcerative colitis or Crohn's disease in human patients. A product comprising an antibody having binding specificity to human α4β7 integrin. The composition contains a dose of 300 mg, and is administered to human patients every 4 weeks or every 8 weeks. The antibody is characterized by the complementarity-determining region 1 (CDR1) shown in SEQ ID NO: 8, and the C shown in SEQ ID NO: 9. It includes a heavy chain variable region including DR2 and CDR3 shown in SEQ ID NO: 10, and SEQ ID NO: 11 Light chains including CDR1 shown, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 13. A composition including a variable region, [2] A set of agents for inducing clinical remission of ulcerative colitis or Crohn's disease in human patients A product comprising an antibody having binding specificity to human α4β7 integrin. The composition contains a dose of 300 mg, and the composition is used to administer the initial dose of the antibody to a human patient. , administering a second dose of the antibody two weeks after the initial dose, and 6 weeks after the initial dose. The antibody is administered according to a dosing regimen that includes administering a third dose of the antibody after a week, and the antibody , complementarity determination region 1 (CDR1) shown in sequence number 8, CDR2 shown in sequence number 9 and sequence It includes a heavy chain variable region including CDR3 shown in number 10, and CDR1 shown in sequence number 11. Includes a light chain variable region including CDR2 shown in Sequence ID No. 12 and CDR3 shown in Sequence ID No. 13. , composition, [3] The ulcerative colitis is moderate to severe active ulcerative colitis, [1] or [2] The treatment methods described in ] [4] Crohn's disease is moderate to severe active Crohn's disease, as described in [1] or [2]. The composition listed, [5] Human patients lacked or lost an appropriate response to TNFα antagonists. The composition described in any of [1] to [4], which was not tolerable therefor [6] A composition for treating ulcerative colitis or Crohn's disease in human patients, The composition comprises an antibody having binding specificity to human α4β7 integrin, and the antibody This includes the complementarity determination region 1 (CDR1) shown in Sequence ID 8, CDR2 shown in Sequence ID 9, and the distribution It includes a heavy chain variable region containing CDR3 shown in column number 10, and CDR1 shown in sequence number 11. , including the light chain variable region which includes CDR2 shown in SEQ ID NO: 12 and CDR3 shown in SEQ ID NO: 13. Furthermore, human patients lacked or lost an appropriate response to TNFα antagonists. , or compositions that were not acceptable thereto [7] The antibody has a heavy chain variable region sequence of amino acids 20-140 of SEQ ID NO: 2 and SEQ ID NO: 4 The combination described in any of [1] to [6], which includes a light chain variable region sequence of amino acids 20 to 131. finished product, [8] The composition according to any one of [1] to [7], wherein the antibody is vedolizumab. [9] The antibody is administered intravenously to a human patient in any of the compositions described in [1] to [8]. ,

[10] The antibody is administered to a human patient as an intravenous infusion over approximately 30 minutes, according to the composition described in [9]. thing Regarding. [Effects of the Invention]

[0039] The present invention may provide formulations for anti-α4β7 antibodies. [Brief explanation of the drawing]

[0040] [Figure 1A]This figure shows the nucleotide sequence (SEQ ID NO: 1) encoding the heavy chain of humanized anti-α4β7 immunoglobulin and the putative amino acid sequence of the heavy chain (SEQ ID NO: 2). The nucleotide sequence includes the cloning site (lowercase), the Kossack sequence (uppercase, nucleotides 18-23 of SEQ ID NO: 1), and the 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] This figure shows the nucleotide sequence (SEQ ID NO: 1) encoding the heavy chain of humanized anti-α4β7 immunoglobulin and the putative amino acid sequence of the heavy chain (SEQ ID NO: 2). The nucleotide sequence includes the cloning site (lowercase), the Kossack sequence (uppercase, nucleotides 18-23 of SEQ ID NO: 1), and the 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 2] This figure shows the nucleotide sequence (SEQ ID NO: 3) encoding the light chain of a humanized immunoglobulin called vedolizumab in this specification, and the putative amino acid sequence of the light chain (SEQ ID NO: 4). The nucleotide sequence includes the cloning site (lowercase), the Kozak sequence (uppercase, nucleotides 18-23 of SEQ ID NO: 3), and the 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. [Figure 3]This figure 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 herein as LDP-02 (SEQ ID NO: 5) (see WO 98 / 06248 and Feagan et al., N. Eng. J. Med. 352:2499-2507 (2005) for LDP-02). 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] (A) This figure shows a sequence comparison of the amino acid sequences of the typical human κ light chain constant region (SEQ ID NO: 6) and the typical mouse κ light chain constant region (SEQ ID NO: 7). The 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 constant region of the human κ light chain, while the 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 constant region of the mouse κ light chain. [Figure 5] This figure shows a map of the vector pLKTOK38D (also known as pTOK38MLN02-TV), which encodes the humanized heavy and light chains of MLN02 and is suitable for vedolizumab production in CHO cells (see U.S. Patent Application Publication No. 2004 / 0033561A1 disclosing pLKTOK38; pLKTOK38D is a variant of pLKTOK38 in which the restriction site shown in the map is adjacent to the sequence encoding the light chain variable region). [Figure 6A] This figure shows a predictive model for changes in monomer ratio, aggregate ratio, and major isoform ratio of an anti-α4β7 lyophilized formulation. The model is based on a statistical analysis of the data presented in Example 1. The center line shows the results of the predictive model, and the outer lines show the 95% confidence limits for the predictive model. [Figure 6B]This figure shows alternative models based on statistical analysis of 40°C data from Tables 1-3, where the input factors are pH, sugar:protein molar ratio, and arginine:protein molar ratio. The center line shows the results of the predictive model, and the outer lines show the 95% confidence intervals for the predictive model. [Figure 7] This figure shows the amino acid sequences of the κ light chain variable region (A) and the human 21 / 28' CL heavy chain variable region (B) of a mature human GM607'CL antibody. [Figure 8] This graph shows how solids and loads affect drying time (the numbers on the line represent the drying time in minutes). [Figure 9] This graph shows that vedolizumab did not delay the onset of clinical symptoms of experimental autoimmune encephalomyelitis (EAE) compared to the placebo control. Natalizumab significantly delayed the onset of clinical symptoms of EAE (p<0.05) compared to the placebo control. [Modes for carrying out the invention]

[0041] The present invention relates to a formulation containing an anti-α4β7 antibody. The formulation contains a non-reducing sugar, an anti-α4β7 antibody, and It may also be a mixture containing at least one free amino acid, a non-reducing sugar and an anti-α4β7 anti- The molar ratio (moles:moles) of the body exceeds 600 reducing sugars to 1 anti-α4β7 antibody. The formulation is solid. It can be in solid or liquid form.

[0042] definition The term "pharmaceutical preparation" refers to a form of anti-α4 antibody that enables the biological activity of an antibody that should be effective. It contains β7 antibodies and additional substances that are unacceptably toxic to the subjects to whom the formulation is administered. This refers to a preparation that does not contain any of the ingredients.

[0043] A "stable" formulation is one in which the antibodies within it maintain their physical stability and / or chemical stability during storage. It substantially retains its inherent stability and / or its biological activity. In one embodiment, The agent, during storage, maintains its physical and chemical stability as well as its biological activity. Keep in storage. The storage period is generally selected based on the intended expiration date of the formulation. Various analytical methods for measuring the stability of the substance are available in this technology, for example, Pept 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 This is outlined in Ivery Rev.10:29-90 (1993).

[0044] "Deamidated" monoclonal antibodies contain one or more asparagine molecules or their glutamates. The n residue is derivatized with aspartic acid or isoaspartic acid.

[0045] Antibodies that are "sensitive to deamidation" are those that are known to be easily deamidated by one or more antibodies. It contains residues.

[0046] Antibodies that are "oxidation-sensitive" contain one or more residues that are known to be easily oxidized. That is the case.

[0047] Antibodies that are "sensitive to agglutination" are particularly susceptible to freezing, heating, drying, reconstitution, and / or stirring. It is known to aggregate with other antibody molecules.

[0048] Antibodies that are "sensitive to fragmentation" can, for example, be cleaved into two or more fragments at their hinge region. It is known that this will happen.

[0049] By "reducing deamidation, oxidation, aggregation, or fragmentation," different pH levels or different pH levels Compared to monoclonal antibodies formulated with a buffer, it inhibits deamidation, aggregation, or fragmentation. To increase or reduce the amount (for example, 80%, 60%, 50%, 40%, 30%, 20%) It is intended to mean (or 10%).

[0050] "Aggregates," "SEC aggregates," or "soluble aggregates" are formed by covalent bonds, ionic bonds, or sparse bonds. They associate together through aqueous interactions to form even larger protein bodies. It is an antibody protein and / or fragment with a size of 10 or less.

[0051] "Insoluble aggregates" or "particles" are formed through covalent, ionic, or hydrophobic interactions. More than 10 antibody proteins and that associate together to form an even larger protein body. / or fragments.

[0052] When used herein, the "biological activity" of a monoclonal antibody refers to its ability to bind to an antigen and test This refers to the ability of antibodies to produce measurable biological reactions that can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

[0053] The cell surface molecule, "α4β7 integrin" or "α4β7", is an α4 chain (CD49D It is a heterodimer of ITGA4 and β7 (ITGB7). Each strand is an alternative integrand. It forms a heterodimer with the phosphorus chain, and is α4β1 or α E It can form β7. Human The α4 and β7 genes (GenBank (National Center for Biotechnology Information, Be thesda, MD), RefSeq acceptance numbers NM_000885 and NM_00088 respectively 9 refers to B and T lymphocytes, especially memory CD4 +It is expressed by lymphocytes. Numerous INT Grin α4β7 can normally exist in either a resting or active state. Ligands for α4β7 include: Vascular adhesion molecules (VCAM), fibronectin and mucosal adrenin (MAdCAM), (For example, MAdCAM-1)) is one such example.

[0054] When used herein, human immunoglobulins having "binding specificity to the α4β7 complex" Phosphorus or its antigen-binding fragments bind to α4β7, but not to α4β1 or αEβ7. stomach.

[0055] When used herein, an “isotonic” preparation has substantially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of approximately 250-350 mOsm. Isotonicity is, for example, For example, it can be measured using a vapor pressure type or ice freezing type osmometer.

[0056] When used herein, "buffering agent" refers to the pH due to the action of its acid / base compound. This refers to a buffer that is resistant to changes. The buffering agent may be present in the liquid or solid formulation of the present invention. The buffering agent of the present invention adjusts the pH of the formulation to approximately 5.0 to approximately 7.5, approximately 5.5 to approximately 7.5, and approximately 6. Adjust the pH to approximately 0.0 to 6.5, or approximately 6.3. In one embodiment, the range is 5.0 to 7.5. Examples of buffering agents that would control pH in the environment include acetates, succinates, and glucons. Histidine, citrate, phosphate, maleate, cacodilate, 2-[N-morpho [Lino] Ethanesulfonic acid (MES), bis(2-hydroxyethyl) iminotris[Hydro [Loxymethyl]methane (bis-tris), N-[2-acetamido]-2-iminodiacetic acid Examples include (ADA), glycylglycine, and other organic acid buffers. In another embodiment, this The buffering agent in the specification is histidine or citrate.

[0057] Histidine buffer is a buffer solution containing histidine ions. Examples of histidine buffer solutions. Examples include solutions of histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate. Histidine buffer or histidine / HCl buffer has a pH of approximately 5.5 to 6.5. The pH is between approximately 6.1 and 6.5, or approximately 6.3.

[0058] In this specification, "sugars" refers to monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, General formula (CH2O) including non-reducing sugars n Compounds having and derivatives thereof. For example, sugars include glucose, sucrose, trehalose, lactose, and fructose. Toxol, maltose, dextran, erythritol, glycerol, arabitol, cyanoacrylate Litol, sorbitol, mannitol, melibiose, meriditose, raffinose, Mannotrious, stachyous, maltose, lactulose, maltose, glucitous Examples include sugars such as maltitol, lactitol, and isomaltulose. Sugars can be frozen. It can be a protective agent. In another embodiment, sugars are non-reducing disaccharides as used herein, even It is sucrose.

[0059] In this specification, "surfactant" refers to an agent that reduces the surface tension of a liquid. It can be a nonionic surfactant. In one embodiment, an example of a surfactant is poly Solvate (polyoxyethylene sorbitan monolaurate, for example, polysorbate) 20 and polysorbate 80); TRITON (t-octylphenoxypolyethoxy) Tanol, a nonionic surfactant, manufactured by Dow Chemical Company in Midland, Michigan. Incarbide subsidiary; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; Sodium octyl glycoside; lauryl-, myristyl-, linoleyl- or stearyl L-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sacro Syn; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, Cocaamidopropyl-, Linoleamidopropyl-, Myristoamidopropyl-, Palmido Propyl-, or isostearmidopropyl-betaine (for example, lauroamide pro Pills; myristoamidopropyl-, palmidopropyl-, or isostearamidopropyl Pyr-dimethylamine; sodium methylcocoyl- or disodium methyloleyl- Tartaric acid; sorbitan monopalmitate; and MONAQUAT series (New Jersey) Mona Industries, Patterson, State; Polyethyl glycol (PEG), Repropylene glycol (PPG) and poloxyethylene and poloxypropylene glycol Examples include copolymers of ru (for example, Pluronics / poloxamer, PF68, etc.). In another embodiment, the surfactant is polysorbate 80.

[0060] In this specification, the term "antibody" is used in its broadest sense, encompassing human antibodies, humanized antibodies, and non-human antibodies. This includes antibodies derived from various species, as well as recombinant antigen-binding forms such as monofunctional antibodies and bifunctional antibodies. full-length monoclonal antibodies, immunoglobulins, polyclonal antibodies, for example, Each is formed from at least two full-length antibodies against different antigens or epitopes. Deviant antibodies (e.g., bispecific antibodies), and dAbs, scFv, Fab, F(a b) Specifically include all individual antigen-binding fragments, including '2' and 'Fab'.

[0061] The molar amounts and molar ratios of anti-α4β7 antibodies to other excipients described herein are determined by the antibody This is calculated based on the assumption of an approximate molecular weight of approximately 150,000 Daltons. The molecular weight of an antibody is determined by its amino acid composition and post-transcriptional modifications, for example, by the molecular weight of the antibody expression molecule. The molecular weight of the antibody can vary by strain, from 150,000 daltons. It could be within ±5% of 000 Daltons.

[0062] The term "human antibody" refers to transgenic mice that possess human immunoglobulin genes. For example, XENOMOUSE genetically engineered mice (California, France) ·BR>c Tower G Abgenix), HUMAB-MOUSE (registered trademark), KIRIN T C MOUSE® transchromosome mouse, KMMOUSE® registered trademark (New Jersey) Human germ cell immunoglobulins, such as antibodies derived from MEDAREX in Princeton, State Robulin sequence, human phage display library, human myeloma cells or human B cells It contains antibodies with the derived sequence.

[0063] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous group of antibodies. This refers to antibodies obtained from, for example, variants that generally exist in small populations. Excluding possible variants that may occur during the production of such monoclonal antibodies, the population is composed of The individual antibodies are identical and / or bind to the same epitope. Usually, different antibodies In contrast to polyclonal antibody preparations containing different antibodies directed towards a specific epitope. Each monoclonal antibody is directed to a single determinant on the antigen. "Ru" refers to the characteristics of antibodies obtained from a substantially homogeneous population of antibodies. This should not be interpreted as requiring antibody preparation by a specific method. For example, this The monoclonal antibody used in accordance with the invention is described by Kohler et al., Nature, 256: It can also be produced by the hybridoma method first described by 495 (1975). By or by recombinant DNA method (see, for example, U.S. Patent No. 4,816,567) It may be produced by [method omitted]. "Monoclonal antibodies" are, for example, Clackson et al., Na ture, 352:624-628 (1991) and Marks et al., J.Mol.Bio Using the technique described in l., 222:581-597 (1991), phage antibody lysate It may be isolated from Brari.

[0064] Specifically, the monoclonal antibodies described herein have a heavy chain and / or a portion of the light chain that are specific to a particular species. The corresponding sequence in antibodies derived from or belonging to a specific class or subclass of antibodies. It is identical or homologous to one other antibody, while the rest of the chain originates from a different species or is a different class of antibody. Alternatively, the sequence is identical or homologous to the corresponding sequence in an antibody belonging to the subclass. "Mela" antibodies, and similarly fragments of such antibodies, as long as they exhibit the desired biological activity, contain (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.) USA, 81:6851-6855 (1984). The chimeric antibodies described herein are for non-human primates (e.g., The variable domain antigen-binding sequence derived from Old World monkeys, apes, etc., and the human constant region Contains "primate-like" antibodies, including those in the row.

[0065] The "antigen-binding fragment" of humanized immunoglobulin prepared by the formulation of the present invention is anti-α4β7 anti It includes at least the variable region of the body's heavy chain and / or light chain. For example, the antigenic region of vedolizumab. The composite fragment contains amino acid residues 20-131 of the humanized light chain sequence of Sequence ID No. 4. Examples of original binding fragments include Fab fragments and Fab' fragments of humanized immunoglobulins known in this technology. Examples include fragments, scFv, and F(ab')2 fragments. Anti-humanized immunoglobulin of the present invention The original binding fragment can be produced by enzymatic cleavage or recombination. For example, papaya Fab fragments or F(ab')2 fragments are generated using pepsin cleavage or pepsin cleavage, respectively. This is possible by using 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. For example, the F(ab')2 fragment. Design a recombinant construct to encode the heavy chain, specifically the CH1 domain and hinge region of the heavy chain. It can include a DNA sequence. In one embodiment, the antigen-binding fragment is α4β7 INT Grin has one or more of its ligands (for example, mucosal addressin MAdCAM (for example, M Inhibits AdCAM1 from binding to fibronectin.

[0066] The papain digestion of antibodies involves fragments called "Fab" fragments, each containing a single antigen-binding site. Two identical antigen-binding fragments, and the remaining "Fc" whose name reflects its ability to easily crystallize. This produces a fragment. Pepsin treatment results in a fragment that still has two antigen-binding sites and still binds to the antigen. A cross-linkable F(ab')2 fragment is obtained.

[0067] "Fv" is a non-covalent association consisting of one heavy chain variable domain and one light chain variable domain. It is an antibody fragment composed of multiple molecules.

[0068] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment is a heavy chain CH containing one or more cysteine ​​derived from the hinge region of the antibody. It differs from Fab due to the addition of two or three residues at the carboxyl terminus of one domain. '-SH is a Fab that has at least one free thiol group in the cysteine ​​residue of the constant region. This is the symbolic representation of ' in this specification. The F(ab')2 antibody fragment originally had a hi between them. It was created as a pair of Fab' fragments containing discysteine. Other chemical components of the antibody fragment Plipping is also known.

[0069] "Single-chain Fv" or "scFv" antibody fragments are antibodies of V H and V L This includes the domain. These domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide is Furthermore, V allows scFv to form the desired surface for antigen binding. H and V L domain Includes polypeptide linkers between them. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antib odies,vol.113,Rosenburg and Moore eds.,S pringer-Verlag, New York, pp.269-315(1994) See below.

[0070] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a variable heavy H -chain domain (V L ) connected to a variable light-chain domain (V L ) by a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains on the other chain to create two H antigen-binding sites. Bispecific antibodies are described more fully, for example, in EP 404,097; WO 93 / 111 61; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 9 0:6444-6448 (1993).

[0071] A "full-length antibody" includes an antigen-binding variable region together with a light-chain constant domain (C L ) and heavy-chain constant domains C H1 , C H2 , H3 C H3 and C . The constant domains may be the constant domains of the native sequence (e.g., the constant domains of the human native sequence) or variants of their amino acid sequences . In one aspect, the full-length antibody has one or more effector functions.

[0072] An "amino acid sequence variant" antibody, as used herein, is an antibody having an amino acid sequence different from that of the major species antibody. Typically, the amino acid sequence variant will have at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95 % homology with the major species antibody. The amino acid sequence variant will have substitutions, deletions and / or additions at particular positions within and / or adjacent to the amino acid range of the major species antibody, but will have antigen-binding activity ​It retains. Mutations in the constant region of the antibody sequence are more significant than mutations in the variable region in the antigen-binding region. The impact on the position is minimal. In the variable region, amino acid sequence variants are less common in major species antibodies. Both are approximately 90% homology, at least approximately 95% homology, at least approximately 97% homology, and at least They are approximately 98% homologous, or at least approximately 99% homologous.

[0073] "Homologousity" involves arranging sequences, introducing gaps where necessary, and achieving the highest proportion of homology. After that, it is defined as the ratio of residues in identical amino acid sequence variants. Sequence comparison The method and computer programs for this are well known in the art.

[0074] "Therapeutic monoclonal antibodies" are antibodies used in treatments for human subjects. The therapeutic monoclonal antibodies disclosed in this document include anti-α4β7 antibodies.

[0075] In this specification, a "glycosylated mutant" antibody is defined as one or more carbohydrate segments linked to a major species antibody. This is an antibody that has one or more carbohydrate moieties linked to it, which are different from the original molecule. (Glycosylation mutation) Examples of bodies in this specification include G1 or G instead of G0 oligosaccharide linked to the Fc region. Antibodies containing two oligosaccharides, and antibodies having one or two carbohydrate moieties linked to the light chain of one or two of these oligosaccharides. , antibodies without carbohydrates linked to one or two heavy chains of the antibody, etc., and sets of glycosylated variants A possible combination is mentioned.

[0076] The "effector function" of an antibody is the Fc region of the antibody (the Fc region of the natural sequence or amino acids). This refers to the biological activity caused by the Fc region of sequence variants. Examples include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, and antibody-dependent cell-mediated cytotoxicity. Cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors, BC) Examples include downward adjustment of R).

[0077] Based on the amino acid sequence of the heavy chain's constant domain, full-length antibodies are divided into different "classes." To be rejected. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG and There are IgM cells, and some of these are further divided into subclasses (isotypes), for example, IgG 1. Can be divided into IgG2, IgG3, IgG4, IgA, and IgA2. Different antibodies The heavy chain constant domains corresponding to each class are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of various classes of globulins are well known.

[0078] The "light chain" of antibodies derived from vertebrate species is based on the amino acid sequence of its constant domain. It is assigned to one of two distinct types called κ or λ.

[0079] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to Fc receptor (FcR) Nonspecific cytotoxic cells that express (e.g., natural killer (NK) cells, phosphatase) (Mesophylls and macrophages) recognize antibodies bound to target cells, and then the target cells are lysed. This refers to a cell-mediated reaction that triggers ADCC. NK cells are the main cells involved in ADCC. While only FcγRIII is expressed, monocytes express FcγRI, FcγRII, and FcγRII It expresses I. FcR expression on hematopoietic cells is observed in Ravetch and Kinet, An Table 3 on page 464 of nu.Rev.Immunol9:457-92(1991) It is summarized. To evaluate the ADCC activity of the molecule, U.S. Patent No. 5,500,36 Perform an in vitro ADCC assay as described in No. 2 or No. 5,821,337. It is also acceptable to use peripheral blood mononuclear cells (PBMs) as effector cells useful for such assays. C) and natural killer (NK) cells are examples. Alternatively or further, the molecule ADCC activity is described, for example, in Clynes et al., PNAS(USA)95:652-656. It may also be evaluated in vivo using animal models such as those disclosed in (1998).

[0080] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of an antibody. It is used. In one embodiment, FcR is a human FcR of the natural sequence. In another embodiment, F cR is a receptor that binds to IgG antibodies (gamma receptor), and allele variants and these FcγRI, FcγRII, and FcγRII include alternatively spliced ​​forms of the receptor. It includes subclass II. FcγRII receptors differ mainly in their cytoplasmic domains. FcγRIIA ("active receptor") and FcγRIIB have similar amino acid sequences. (An "inhibitory receptor") is one example. The active receptor FcγRIIA has a cytoplasmic domain It contains an activation motif (ITAM) based on the immune receptor tyrosine. Somatic FcγRIIB has an inhibitory motif based on the immune receptor tyrosine in its cytoplasmic domain. (ITIM) contains (M. Daeron, Annu. Rev. Immunol. 15 See the overview in 203-234 (1997). FcR is Ravetch and K inet,Annu.Rev.Immunol9:457-92(1991);Cape l, Immunomethods 4:25-34 (1994); and de Haa This was outlined in 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 FcRn, a neonatal receptor 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)).

[0081] When used herein, the term "hypervariable region" refers to the amino acids of an antibody involved in antigen binding. This refers to a residue. The hypervariable region is generally called the "complementarity-determining region" or "CDR" (for example, light chain variable Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the mutational region 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)) Derived amino acid residues and / or "hypervariable loops" (e.g., residues in the light chain variable region) Base 26-32 (L1), 50-52 (L2), and 91-96 (L3) and heavy chain variable region Residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in Chothi Contains residues derived from a and Lesk J. Mol. Biol. 196:901-917 (1987). The residues in the "- region" or "FR" are other than the residues in the hypervariable region as defined herein. These are residues of the variable domains. The hypervariable region or its CDR one antibody chain or another chain or The antigen-binding specificity of the (complex) antibody or binding protein obtained by transferring it to another protein is then determined. Grant.

[0082] Humanized forms of non-human (e.g., rodent) antibodies are derived from non-human immunoglobulins. It is a chimeric antibody containing a small number of sequences. For most of the parts, humanized antibodies are human. The immunoglobulin (recipient) is derived from the hypervariable region of the recipient, A product possessing the desired specificity, affinity, and capabilities, for example, from mice, rats, rabbits, or non-humans. It is replaced by residues (donor antibodies) derived from the hypervariable region of non-human species such as primates. In some cases, residues in the human immunoglobulin framework (FR) correspond to non-human residues. It is replaced by a residue. Furthermore, humanized antibodies are found in recipient antibodies or donor antibodies. It may contain residues that are not present. These modifications are performed to further improve the performance of the antibody. Generally, human Antibodies contain virtually all of at least one, usually two, variable domains, and are highly variable. All or substantially all of the globulins are equivalent to those of non-human immunoglobulins, and all of the FR are equivalent to those of non-human immunoglobulins. Or, virtually all, are those of human immunoglobulins. Humanized antibodies are optional, and immunoglobulins It will include at least a portion of the constant region (Fc) of brin, which is typically that of human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525. 1986); Riechmann et al., Nature, 332:323-329 (1988) ); and Presta, Curr. Op. Struct. Biol. 2:593-596 See (1992).

[0083] "Affinity-matured" antibodies have a higher affinity for the antigen compared to parental antibodies that do not undergo these changes. It has one or more changes in one or more hypervariable regions to improve it. Embodiment 1 In one case, affinity-mature antibodies have nanomolar or even picomolar affinity for the target antigen. It will. Affinity-matured antibodies are produced by procedures known in this technology. Mark s et al., Bio / Technology, 10:779-783 (1992) describes VH and VL This describes affinity maturation through substitution of CDR and / or framework residues. Artificial mutagenesis was investigated 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 As described in 92).

[0084] "Isolated" antibodies are identified and isolated from components of their natural environment and / or It is recovered. In certain embodiments, the antibody is (1) measured by the Lowry method. When exceeding 95% by weight of protein, or exceeding 99% by weight, (2) spin-cap The use of a top sequencer allows for the extraction of at least 15 residues of the N-terminal or internal amino acid sequence. To a sufficient extent to obtain, or (3) under reducing conditions using Coomassie blue staining or silver staining Alternatively, it will be purified to homogeneity by SDS-PAGE under non-reducing conditions. The antibodies produced lack at least one component of the antibody's natural environment, therefore, within recombinant cells... It contains antibodies produced in situ. However, normally, isolated antibodies are produced at least once. It will likely be prepared during the purification process.

[0085] "Treatment" refers to both therapeutic measures and preventive or protective measures. This includes conditions where the disease or its recurrence should be prevented, similar to conditions where the disease already exists. Therefore, patients treated in this specification may be diagnosed with a disease, or have a disease. They may be susceptible to or prone to the disease. The terms “patient” and “subject” are used herein. They are used interchangeably.

[0086] The antibodies being formulated are substantially pure, and preferably substantially homogeneous (without contamination). (Does not contain protein, etc.). A "substantially pure" antibody is defined as having a total protein content in the composition. A set containing at least about 90% by weight, at least about 95 or 97% by weight of antibodies, based on quantity. It means a finished product. A "substantially homogeneous" antibody is at least based on the total weight of the protein. Approximately 99% by weight of the protein is a specific antibody, such as an anti-α4β7 antibody. It means a composition.

[0087] When "clinical remission" is used herein in relation to ulcerative colitis, it means complete remission. This refers to a Mayo score of 2 or less, with no individual sub-scores exceeding 1. "Clinical remission" of Roon's disease refers to a CDAI score of 150 or less.

[0088] When "clinical response" is used herein in relation to ulcerative colitis, it means 1 point or less. Complete May A score of 3 or higher or a 30% decrease from baseline (complete Mayo score in outpatient settings) If you did not perform the test, you must have a partial Mayo score of 2 or more points or 2 points from the baseline. This refers to a rate of 5% or higher. "Clinical response" is used herein in relation to Crohn's disease. This refers to a decrease of 70 points or more in the CDAI score from baseline (week 0).

[0089] When "mucosal healing" is used herein in relation to ulcerative colitis, it refers to a score of 1 or less. This refers to the endoscopic subscore.

[0090] As used herein, “treatment failure” refers to the treatment of ulcerative colitis or Crohn’s disease. This refers to the worsening of a disease, the need for emergency treatment, or surgical intervention. Emergency treatment refers to new or untreated conditions. New medications or treatments needed to resolve the symptoms of ulcerative colitis or Crohn's disease This involves increasing the dosage of line-administered medications (other than antidiarrheal drugs for controlling chronic diarrhea).

[0091] formulation As described herein, anti-α4β7 antibodies reduce excess non-reducing sugars (on a molar basis). When the formulation is dried, for example, lyophilized, it has been found to be highly stable. In particular, the ratio (moles:moles) of non-reducing sugars to anti-α4β7 antibodies exceeds 600:1. This specification indicates that the lyophilized formulation is stable for at least two years.

[0092] The present invention provides a stable anti-α4β7 antibody preparation in a first embodiment. In one embodiment, The agent comprises a buffer, at least one stabilizer, and an anti-α4β7 antibody. In one embodiment, drying The preparation contains one or more non-reducing sugars and an anti-α4β7 antibody, and the ratio of non-reducing sugars to anti-α4β7 antibodies (moles) The ratio (moles) is greater than 600:1. The formulation also contains one or more free amino acids. Mino acids can act as buffers. In one embodiment, one or more amino acids are stable. It can act as an agent. The formulation optionally further comprises at least one surfactant. Obtain. In one embodiment, the formulation is dried, for example, lyophilized. The antibody may be a full-length antibody, or Fab, Fv, scFv, Fab', or F(a b')2 may be the antigen-binding fragment.

[0093] The formulation may contain a desired non-reducing sugar. In one embodiment, the formulation may include Examples of non-reducing sugars include mannitol, sorbitol, sucrose, and trehalose. S, raffinose, stachyose, melegitose, dextran, maltitol, lactin Examples include tol, isomaltulose, palatinite, and combinations thereof. Another embodiment Non-reducing sugars include sucrose, trehalose, mannitol, and sorbitol. The absolute amount of non-reducing sugar in a formulation is not important, but the ratio (moles) of non-reducing sugar to anti-α4β7 antibody is important. The ratio (moles) of non-reducing sugar to anti-α4β7 antibody (moles) exceeds 400:1. In another embodiment, the ratio (moles) of non-reducing sugar to anti-α4β7 antibody is greater than 400:1. The mole ratio is at least approximately 600:1; at least approximately 625:1; at least approximately 650:1 ; at least approximately 675:1, at least approximately 700:1; at least approximately 750:1, less Both are approximately 800:1, at least approximately 1000:1, at least approximately 1200:1, at least Approximately 1400:1, at least approximately 1500:1, at least approximately 1600:1, at least approximately 1700:1, at least about 1800:1, at least about 1900:1, or at least The ratio is approximately 2000:1. Generally, for example, during freeze-drying and / or drying and reconstitution, It is desirable to have non-reducing sugars present in an amount that reduces the formation of soluble aggregates, such as those that form in soluble aggregates. It seems that a ratio (moles:moles) of non-reducing sugar to anti-α4β7 antibody higher than approximately 730:1 is frozen. In a dry state, a slightly reduced amount of soluble aggregates may form. The sugar:protein weight ratio is 1.5 :Can be made greater than 1(w / w). In another embodiment, liquid (for example, before drying or The concentration of non-reducing sugars in the (reconstituted) formulation is approximately 10 mM to 1 M, for example, approximately 60 mM M ~ about 600mM, about 100mM - 450mM, about 200mM - about 350mM, about 250 The range is approximately mM to 325 mM, and approximately 275 mM to 300 mM. In one embodiment, The amount of non-reducing sugars in dried (e.g., freeze-dried) formulations is approximately 40% to approximately 70% (dried). It is within the w / w range of the formulation. In another embodiment, in a dried (e.g., lyophilized) formulation, The amount of non-reducing sugars is approximately 40% to 60%, 45% to 55%, or 51% (w / w). It is within the range. In other embodiments, the amount of protein in the dried formulation is approximately 31% (w / of the dried formulation). w) or if the mass ratio of non-reducing sugars to proteins exceeds 1.6:1, drying (even if However, the amount of non-reducing sugars in the freeze-dried formulation exceeds approximately 51% (w / w of the dried formulation). Furthermore, in yet another aspect, sucrose is a non-reducing sugar for use in pharmaceutical preparations.

[0094] The formulation may contain the desired free amino acid, which may be in L-form, D-form, or These can be desired mixtures of these forms. In one embodiment, the formulation may contain free The amino acids involved include, for example, histidine, alanine, arginine, glycine, and glutamic acid. Phosphoric acid, serine, lysine, tryptophan, valine, cysteine, and combinations thereof include. Some amino acids can stabilize proteins against degradation during production, drying, lyophilization, and / or storage, for example, through hydrogen bonding, salt bridging, antioxidant properties, or hydrophobic interactions, or by excluding them from the protein surface. Amino acids can act as tonicity regulators or act to lower the viscosity of the formulation. In another aspect, free amino acids such as histidine and arginine can act as cryoprotectants and lyoprotectants and do not crystallize when lyophilized as a component of the formulation. Free amino acids such as glutamic acid and histidine can act as buffers in aqueous solutions at a pH in the range of 5 - 7.5, either alone or in combination. Furthermore, in yet another aspect, the formulation contains histidine, or histidine and arginine. Furthermore, in yet a further aspect, the concentration of free amino acids for liquid formulations is in the range of about 10 mM to about 0.5 M, for example, about 15 mM to about 300 mM, about 20 mM to about 200 mM, or about 25 mM to about 150 mM, about 50 mM, or about 125 mM. Furthermore, in yet a further aspect, the amount of histidine in a dry (e.g., lyophilized) formulation is in the range of about 1% to about 10% (w / w of the dry formulation) or about 3% to about 6% (w / w). In some embodiments, when the amount of protein is about 31% (w / w of the dry formulation) or the mass ratio of histidine to protein exceeds about 0.15:1 in the dry (e.g., lyophilized) formulation, the amount of histidine in the dry (e.g., lyophilized) formulation exceeds about 4% (w / w of the dry formulation). Furthermore, in yet another aspect, the amount of arginine in a dry (e.g., lyophilized) formulation is about The concentration is in the range of 4% to approximately 20% (w / w of the dry formulation) or approximately 10% to approximately 15% (w / w). In some embodiments, the amount of protein is approximately 31% (w / w of the dry formulation) Or if the mass ratio of arginine to protein exceeds approximately 0.4:1, drying (for example, The amount of arginine in the freeze-dried formulation exceeds approximately 13% (w / w of the dry formulation). In embodiments of amino acid combinations such as stidine and arginine, the total amino acids and antibodies The molar ratio is at least 200:1, approximately 200:1 to approximately 500:1, or at least 400 It can be :1.

[0095] The formulation may optionally further contain at least one surfactant. Embodiment 1 One example of a surfactant that can be included in a formulation is, for example, polysorbate. 20. Polysorbate 80, Poloxamer (Pluronic® and combinations thereof) Combinations are mentioned. If present, surfactants are generally used, for example, in bottling, freezing, During drying, freeze-drying, and / or reconstitution, include an amount that reduces the formation of insoluble antibody aggregates. This can be done, for example, at the interface in the pre-drying (e.g., lyophilized) formulation or the reconstituted formulation. The concentration of the activator is generally about 0.0001% to about 1.0%, about 0.01% to about 0.1%, for example For example, approximately 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, %, or 0.09% (w / v), 0.05%~0.07% or 0.06% (w / v) The amount of surfactant in a dried (e.g., freeze-dried) formulation is generally about 0. 0.1% to approximately 3.0% (w / w), approximately 0.10% to approximately 1.0%, for example, approximately 0.15%. 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, or 0.50% (w / w) In another embodiment, the mole ratio of surfactant to antibody is approximately 1:1. Non-reducing sugar and antibody Under the condition that the ratio (moles:moles) of α4β7 antibodies exceeds approximately 600:1, the anti-α4β7 antibody is The formulation can be present in the desired amount. However, the formulation contains high concentrations of anti-α4β7 anti It may contain a substance. For example, a liquid formulation may contain at least about 10 mg / ml, and less than At least approximately 20 mg / ml, at least approximately 30 mg / ml, at least approximately 40 mg / ml, At least approximately 50 mg / ml, at least approximately 60 ml / ml, at least approximately 70 mg / m³ l, at least about 80 mg / ml, at least about 90 mg / ml, at least about 100 mg / ml g / ml, approximately 40 mg / ml to approximately 80 mg / ml of anti-α4β7 antibody, approximately 60 mg / ml It may contain an anti-α4β7 antibody. The dry formulation (e.g., lyophilized) is by weight, At least about 5%, at least about 10%, at least about 15%, at least about 20%, and less It contains at least 25%, at least 30%, or about 31% or 32% anti-α4β7 antibodies. It is possible to have.

[0096] If desired, the formulation may also contain metal chelating agents and other pharmaceutically acceptable excipients. / or may contain antioxidants. Suitable metal chelating agents include, for example, methylamine. N, ethylenediamine, desferoxamine, trientine, histidine, malate, Phosphonate compounds, for example, etidronic acid, ethylenediaminetetraacetic acid (EDTA), Examples include ethylene glycol tetraacetic acid (EGTA). Suitable antioxidants include, for example, Citric acid, uric acid, ascorbic acid, lipoic acid, glutathione, tocopherol, carotene , including lycopene, cysteine, etc.

[0097] The formulation can be liquid or solid. Liquid formulations are aqueous solutions or aqueous suspensions prepared in a suitable aqueous solvent such as water or an aqueous / organic mixture such as a water / alcohol mixture. It can be a suspension. Liquid formulations can have a pH between about 5.5 and about 7.5, between about 6.0 and about 7.0, between about 6.0 and about 6.5, for example, about 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. Liquid formulations can be stored at room temperature, refrigerated (e.g., 2 - 8°C) or frozen (e.g., -20°C or -80°C). Solid formulations can be prepared by suitable methods, for example, in the form of a cake or powder by adding a cryoprotectant. Solid formulations can be in the form of spheres for oral delivery or films for transdermal delivery, for example, by freeze-drying, spray-drying, air-drying in a film (e.g., for transdermal delivery), mixing with a liquid emulsion and drying to dry the liquid formulation as described herein. When the formulation is a solid formulation, the formulation can contain slightly about 5%, slightly about 4.5%, slightly about 4%, slightly about 3.5%, slightly about 3%, slightly about 2.5%, slightly about 2%, slightly about 1.5%, slightly about 1% moisture, or be substantially anhydrous. Solid formulations can be dissolved, i.e., reconstituted in a suitable medium to become a suitable liquid for administration. Solvents suitable for reconstituting solid formulations include water, isotonic saline, buffers such as phosphate-buffered saline, Ringer's (with lactate or dextrose) solution, essential inorganic media, alcohol / aqueous solutions, dextrose solutions, etc. The amount of the solvent can result in a higher, same or lower concentration of the therapeutic protein than the amount before drying. ​​​​​​​​​ In one embodiment, the concentration of the reconstituted anti-α4β7 antibody is the same as that of the liquid formulation before drying. It is concentration.

[0098] The formulation may be sterile, which is suitable for administration to human subjects before or after preparation of the formulation. This can be achieved by following procedures known to those skilled in the art for producing sterile pharmaceutical formulations. For example, sterilization is performed by filtration through small pores before drying and / or after reconstitution. It can be sterilized as a liquid by means of or exposure to ultraviolet irradiation. (filter) - The pore size is 0.1 μm to 0.2 μm for filtering microorganisms, and for filtering virus particles. The wavelength can be 10-20 nm. Alternatively, or even better, the dry formulation can be For example, sterilization can be achieved by exposure to gamma radiation. In one embodiment, anti-alpha The 4β7 antibody liquid formulation is sterilized by filtration before drying.

[0099] In one embodiment, the formulation is stable during storage. In another embodiment, the formulation is stable in a dry state. It is stable during storage. The stability is maintained before and after formulation, and after storage at the aforementioned temperature. To evaluate the physical, chemical, and / or biological stability of antibodies in formulations. This can be investigated by the physical and / or of the liquid formulation or reconstituted dry powder. Chemical stability is evaluated by assessing aggregate formation (e.g., size exclusion (or gel filtration) chromatography). Graph (SEC), Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MA) LDI-TOF-MS), analytical ultracentrifugation, light scattering methods (optical correlation spectroscopy, dynamic light scattering ((D LS) or multi-angle laser scattering (MALLS), flow-based microscopy imaging, electron Impedance (Coulter) counting method, light obscuration or other liquid particle counting methods, (turbidity By measurement, by density gradient centrifugation and / or visual inspection); cation exchange Lammagraphy (Vlasak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) an (See also Harris et al. J. Chromatogr. B Biomed. Sci. Appl. 752:233-245 (2001)), Isoelectric focusing (IEF), for example, capillary electrophoresis or capillary zone electrophoresis. By evaluating charge heterogeneity using; sequence analysis of amino or carboxyl termini. Mass spectroscopy analysis; of fragmented, intact, and polymers (e.g., dimers, trimers, etc.) SDS-PAGE or SEC analysis for antibody comparison; peptide mapping (e.g., Lipsin or LYS, etc.); various methods including evaluating the biological activity or antigen-binding function of antibodies. Different methods (for example, Analytical Techniques for Biopharmaceutical Development, R (Refer to odriguez-Diaz et al. eds. Informa Healthcare (2005)) Qualitatively and / or This can be quantitatively evaluated. This includes biological activity or anti-α4 binding function, for example, anti-α4. β7 inhibitor Binding to MAdCAM (e.g., MAdCAM-1) or α4β7 integrin MAdCAM expressed in cells (e.g., MAdCAM-1), for example, immobilized M Inhibition of binding to AdCAM (e.g., MAdCAM-1) is available to skilled physicians. It can be evaluated using various techniques (for example, Soler et al., J. Pharmaco See l. Exper. Ther. 330:864-875 (2009).

[0100] The stability of a solid-state formulation can be determined, for example, by examining its crystal structure using X-ray powder diffraction (XRPD). Identifying the antibody structure in the solid state using Fourier transform infrared spectroscopy (FTIR). Evaluating the structure; using differential scanning calorimetry (DSC, for example, to evaluate denaturation) Direct tests such as measuring thermal transitions (melting, glass transition, etc.) in freeze-dried solids, and The Karl fish-fishing test measures the moisture content and indicates chemical instability via hydrolysis. This can be qualitatively influenced by various different methods, including indirect tests such as estimating the likelihood of sex. It can also be evaluated by / or quantitatively. Measuring the moisture content of a dry formulation indicates how the formulation changes This indicates whether the substance undergoes chemical or physical decomposition; higher moisture content leads to further decomposition.

[0101] Stability can be measured at a selected temperature for a selected time. In one embodiment, (For example, freeze-dried) formulations should be stored at approximately 40°C and 75% RH for at least approximately 2-4 weeks. , for at least approximately 2 months, at least approximately 3 months, at least approximately 6 months, at least approximately It remains stable for 9 months, at least about 12 months, or at least about 18 months. Another embodiment The formulation (liquid or dry (e.g., lyophilized)) is suitable for temperatures of approximately 5°C and / or 25°C. And at 60% RH for at least approximately 3 months, at least approximately 6 months, and at least approximately 9 months. , at least approximately 12 months, at least approximately 18 months, at least approximately 24 months, less Both remain stable for approximately 30 months, at least approximately 36 months, or at least approximately 48 months. In another embodiment, the formulation (liquid or dry (e.g., lyophilized)) is slightly reduced in temperature at approximately -20°C. At least approximately 3 months, at least approximately 6 months, at least approximately 9 months, at least approximately 12 months, at least about 18 months, at least about 24 months, at least about 30 months, Stable for at least approximately 36 months, at least approximately 42 months, or at least approximately 48 months Yes. Furthermore, in some embodiments, the liquid formulation can be frozen (for example, to -80°C) and After thawing, for example, after one, two, or three freeze-thaw cycles, it may be stable.

[0102] Instability can manifest as aggregation (for example, non-covalent interactions (caused by hydrophobic or charge interactions)). Covalent soluble aggregation (e.g., disulfide bond rearrangement) and covalent soluble aggregation (e.g., disulfide bond rearrangement). (Displacement), insoluble aggregation (caused by protein denaturation at the liquid / air and liquid-solid interfaces) (causing), deamidation (e.g., deamidation of Asn), oxidation (e.g., Met) Oxidation, isomerization (e.g., isomerization of Asp), denaturation, clipping / hydrolysis / fragmentation (For example, fragmentation of the hinge region), succinimide formation, N-terminal elongation, C-terminal p One or more of the following may be involved: loss of glycosylation, differences in glycosylation, etc.

[0103] Stable formulations can contribute to the low immunogenicity of anti-α4β7 antibodies. α4β7 antibodies can induce human / anti-human antibody (HAHA) reactions in human subjects or patients. Patients who develop an HAHA reaction to anti-α4β7 antibodies may experience adverse events during treatment (for example) For example, it can have a site-injection reaction, or it can rapidly eliminate anti-α4β7 antibodies. This can result in a lower dose than planned due to the treatment. Early trials of anti-α4β7 antibody therapy. The report (Feagen et al. (2005) N. Engl. J. Med. 352:2499-2507) showed that the treated patients The study showed that human anti-human antibodies were generated in 44% of participants by 8 weeks. It was stored as a liquid and did not contain polysorbate.

[0104] In some embodiments, the formulation yields H results compared to less stable formulations. The proportion of AHA-negative patients should be at least 40%, at least 50%, and at least 60% of the patients. It can be increased to %, at least 70%, at least 80%, or at least 90%. ru.

[0105] In some embodiments, the anti-α4β7 antibody preparation contains ≥50% of the major charged isoforms. ≥55% of the major charged isoforms, or 65-70% of the major charged isoforms It possesses. In another embodiment, a stable anti-α4β7 antibody preparation is an acid-charged isophore with an acidity of ≤45%. M, ≤40% acid-charged isoform, ≤30% acid-charged isoform or 22~ It has a 28% acidic charged isoform. In another embodiment, a stable anti-α4β7 antibody The formulations include basic isoforms of ≤25%, basic isoforms of ≤20%, and ≤15%. Basic isoforms, approximately 5% basic isoforms, or approximately 10% basic isoforms It has a form. In one embodiment, a stable anti-α4β7 antibody preparation is, for example, by CEX When measured, it shows ≥55% of the major isoforms, ≤30% of the acidic isoforms, and / or having a basic isoform of ≤20%. In another embodiment, for example, cIEF Therefore, when measured, ≥50% of the major isoforms and ≤45% of the acidic isoforms and It has a basic isoform of ≤10%.

[0106] In some embodiments, the anti-α4β7 antibody dry solid formulation contains ≤10% moisture, ≤5% moisture, or It has a moisture content of <2.5%. The time required for reconstruction is ≤60 minutes, ≤50 minutes, or ≤4 The time is 0 minutes, or ≤30 minutes, or ≤20 minutes.

[0107] monomer content and / or aggregate content in liquid formulations or reconstituted dry formulations (for example) (as dimers, trimers, tetramers, pentamers, oligomers, and even higher-order aggregates) are S EC, MALDI-TOFMS, analytical ultracentrifugation, light scattering methods (DLS or MALLS), This involves nanoscale measurements, such as nanoparticle tracking analysis (NTA, Wiltshire, UK). Measurement can be performed by anoSight. This includes resolution, property analysis, and quantification of aggregates. For example, by using a longer column or along the original analytical SEC column, a second or subsequent column By continuously linking the above SEC columns, the length of the SEC column separation is increased, and light diffusion Numerous methods, including supplementing monomer SEC quantification with randomization, or using NTA This can be achieved by doing so.

[0108] In one embodiment, the anti-α4β7 antibody preparation comprises ≥90% monomeric antibody and ≥95% monomeric antibody. The antibody has a body or 97-99% monomeric antibody. In another embodiment, the anti-α4β7 antibody preparation has Most materials in this context have an average size of ≤20nm, ≤15nm, ≤10nm, or approximately 5-7nm. It has a radius. In one embodiment, the anti-α4β7 antibody preparation is determined by protein analysis to be ≥80% It has a quantity of heavy chains and light chains. In one embodiment, there are ≥90% heavy chains and light chains. In another embodiment, Anti-α4β7 antibody preparations showed aggregates of ≤10%, ≤5%, ≤2.5%, and ≤1 It has 0.5% aggregates, ≤1% aggregates, or ≤0.5% aggregates. In another embodiment, it has anti-α The 4β7 antibody preparation has ≥96% monomers and / or ≤2.5% aggregates. In one embodiment, the anti-α4β7 antibody preparation has ≥99% monomers and / or ≤1% aggregates.

[0109] For example, the particle size of aggregates or undissolved excipients in the reconstituted formulation is obscured by light. Chemical methods (for example, Hach Ultra Analytics (Oregon, Grampus) Liquid particle counting (HIAC) by ) microscope, Coulter counter, or Brigh Digital microfluidics imaging (MFI) by twell (Ottawa, Canada) A method based on flow-based microscopy imaging, or fluid imaging (Maine, Yarmouth). It can be measured using the FLOWCAM® image particle analyzer. In one embodiment, the particle size of the anti-α4β7 antibody preparation is approximately 30 μm, approximately 25 μm, and approximately The particle size is 10 μm, approximately 5 μm, approximately 2 μm, or less than 1 μm. The amount of particles can be produced with antibody preparations. Only this should be kept under control. In one embodiment, the anti-α4β7 antibody preparation is administered in a single dose of ≥10 μg. Having particles less than 6000 of size m and less than 600 of size ≥ 25 μm (United States Pharmacopeia Chp 0.788, light obscuration counting method; half of those amounts by microscopic quantification method). Yet another aspect So, regarding anti-α4β7 antibody preparations, for example, the MFI measurement method at the dose of a reconstituted preparation... The amount of particles per milliliter is approximately 500 to 2000 particles of 2-10 μm per ml. 000 or approximately 1000 to approximately 3000, with approximately 50 to approximately 350 particles of ≥10μm per ml. And the number of particles with a diameter of ≥25 μm per ml is approximately 0 to 50.

[0110] In one embodiment, the anti-α4β7 antibody preparation is approximately 60% to approximately 1% of the reference standard anti-α4β7 antibody. It has a binding affinity of 40%. In one embodiment, the anti-α in the formulation described herein. 4β7 antibody is present at approximately 80% to 120% of the reference standard, for example, in cells (WO98 / 0 It binds to α4β7 on Patent No. 6248 or U.S. Patent No. 7,147,851. Another embodiment So, anti-α4β7 antibody preparations are used in MAdCAM cells that express α4β7 integrin. For example, MAdCAM-1, for example, MAdCAM-Ig chimera (in a reference standard sample) (See also U.S. Patent Application No. 20070122404) The combination to at least 50% It has the ability to inhibit at least 60%.

[0111] As mentioned above, freezing of the formulation is specifically intended in this specification. Therefore, freezing The stability of the formulation during melting can be investigated. As a result, the antibody in liquid formulations can be examined. The body can be stable during the freeze-thaw cycle of the preparation, and for example, antibodies can be frozen 1, 2, 3, 4, 5 times. After the above freeze-thaw cycle, it can remain stable.

[0112] In some embodiments, the formulation contains at least about 50 mg / mL to about 100 mg / mL of antimicrobial agent. α4β7 antibody, buffering agent (e.g., histidine), and at least 9% (w / w) non- It is a liquid formulation containing reducing sugars (for example, sucrose, trehalose, or mannitol). In one embodiment, the formulation contains approximately 50 to approximately 80 mg / ml and approximately 60 mg / ml of anti-α4β7 Antibodies, buffering agents (e.g., histidine), and free amino acids (e.g., arginine) And at least 9% or 10% (w / w) of non-reducing sugars (e.g., sucrose, trehalose) Contains (or mannitol).

[0113] In another embodiment, the formulation contains at least about 60 mg / ml of anti-α4β7 antibody and buffered A drug (e.g., histidine), a free amino acid (e.g., arginine), and at least Also 10% (w / w) of non-reducing sugars (e.g., sucrose, trehalose, or mannitol) ) includes. In such embodiments, the buffer concentration is approximately 15 to approximately 75 mM, and approximately 25 to approximately 65 The concentration is mM or approximately 50 mM. The concentration of free amino acids is approximately 50 to approximately 250 mM, approximately 75 to approximately This is 200 mM, approximately 100-150 mM, or approximately 120 mM.

[0114] In one embodiment, the formulation comprises a non-reducing sugar, an anti-α4β7 antibody, histidine, and arginine. and a dry solid formulation containing a mixture of polysorbate 80 (for example, a freeze-dried formulation) The agent is such that the molar ratio (moles:moles) of non-reducing sugar to anti-α4β7 antibody exceeds 600:1.

[0115] In another embodiment, the formulation comprises a non-reducing sugar, an anti-α4β7 antibody, histidine, and arginine. A dry, solid amorphous formulation containing a mixture of polysorbate 80 (for example, freeze-dried (Dried formulation), and the molar ratio (moles:moles) of non-reducing sugar to anti-α4β7 antibody is 600:1. To surpass.

[0116] In one embodiment, the formulation comprises a non-reducing sugar, an anti-α4β7 antibody, histidine, and arginine. Furthermore, it is a lyophilized preparation containing polysorbate 80, and a non-reducing sugar and an anti-α4β7 antibody. The molar ratio (moles:moles) exceeds 600:1.

[0117] In one embodiment, the formulation comprises a non-reducing sugar, an anti-α4β7 antibody, histidine, and arginine. Furthermore, it is a freeze-dried formulation containing polysorbate 80, and the formulation contains non-reducing sugars and anti-α4 The molar ratio (moles:moles) of β7 antibodies exceeds 600:1, and the arginine and anti-α4 ratio in the formulations The molar ratio (moles:moles) of the β7 antibody exceeds 250:1.

[0118] In one embodiment, the formulation is a liquid formulation containing at least about 60 mg / ml of anti-α4β7 anti- Body, at least 10% (w / v) non-reducing sugars, and at least 1 or more active ingredients of approximately 125 mM. It contains the amino acids.

[0119] In one embodiment, the formulation is a liquid formulation containing at least about 60 mg / ml of anti-α4β7 anti- Body, at least 10% (w / v) non-reducing sugars, and at least 1 or more active ingredients of approximately 175 mM. It contains the amino acids.

[0120] In one embodiment, the formulation is a liquid formulation containing approximately 60 mg / ml to approximately 80 mg / ml of anti-α- It contains 4β7 antibody, a buffering agent, and at least approximately 10% (w / w) of sugar.

[0121] In one embodiment, the formulation is a liquid formulation containing approximately 60 mg / ml to approximately 80 mg / ml of anti-α- It contains 4β7 antibody, histidine, and at least approximately 10% (w / w) sucrose.

[0122] In one embodiment, the formulation is freeze-dried and stored in a single vial as a single dose. The vials should preferably be stored at approximately 2-8°C until administered to the target who needs them. The vials (for example, for a 60 mg / ml dose) contain, for example, 20 or 50 cc. It is possible. The vial contains at least approximately 120 mg, at least approximately 180 mg, and at least Also about 240 mg, at least about 300 mg, at least about 360 mg, at least about 54 It may contain 0 mg or at least about 900 mg of anti-α4β7 antibody. In one embodiment, The vial contains approximately 300 mg of anti-α4β7 antibody.

[0123] Remington:The Science and Practice of P Harmacy, 21st edition, Hendrickson, R.Ed. (2005) One or more other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those found in the formulation, are used. It may be included in the formulation provided that it does not adversely affect the desired characteristics. Acceptable carriers, The excipients or stabilizers are non-toxic to the recipient at the dosage and concentration used, and In addition, additional buffering agents; cosolvents; antioxidants including ascorbic acid and methionine; ED Chelating agents such as TA; metal complexes (e.g., Zn / protein complexes); polyesters Examples include biodegradable polymers such as; and / or salt-forming counterions such as sodium. ru.

[0124] α4β7 antibody Suitable anti-α4β7 antibodies for use in pharmaceutical formulations include, for example, complete human antibodies, mouse antibodies, Antibodies from desired sources such as rabbit antibodies, and, for example, chimeric antibodies, humanized antibodies. This includes desired manipulated antibodies such as Fab, Fv, scFv, Fa Antigen-binding fragments of any of these types of antibodies, such as the b' and F(ab')2 fragments, are also included. It is suitable for use in pharmaceutical formulations.

[0125] Anti-α4β7 antibodies target epitopes on the α4 chain (e.g., humanized MAb21.6 (Bendig) et al., US Pat. No. 5,840,299)), Epitope on β7 chain (FIB504 or humanized Derivatives (e.g., Fong et al., U.S. Patent No. 7,528,236), or α4 chain and β It can bind to a combination epitope formed by the association of seven chains. Embodiment 1 In one case, the antibody binds to a combination epitope on the α4β7 complex, but the chains associate with each other. Unless this occurs, it will not bind to epitopes on the α4 or β7 chain. Association with ntegrin brings the residues present on both strands containing the epitope into close proximity. By, or on one of the chains, for example, the α4 integrin chain or the β7 integrin chain. Antibody binding occurs in the absence of a suitable integrin partner or in the absence of integrin activation. By stereochemically exposing antibody binding sites that are inaccessible, combined epitome It is possible to create a p. In another embodiment, the anti-α4β7 antibody is α4 integrin chain and Because it binds to both sides of the β7 integrin chain, it is specific to the α4β7 integrin complex. Yes, such antibodies can bind α4β7, but for example, they can bind α4β1. They cannot be combined, and / or α E β7 cannot be bound. In another embodiment, 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) is the same or substantially the same. It binds to the pitope. Mouse ACT that produces Act-1 monoclonal antibody. -1 Hybridoma cell lines were defined under the provisions of the Budapest Convention of August 22, 2001. 40 M Landsdowne Street, Cambridge, Massachusetts, USA 02139 In order to benefit Illennium Pharmaceuticals, 110-2209 Virginia, Manassas Boulevard University, 10801 American University It was deposited with the Ipculture Collection under receipt number PTA-3663. In another form, The anti-α4β7 antibody is provided in the CDR provided in U.S. Patent Application Publication No. 2010 / 0254975. This is a human antibody or α4β7-binding protein using [a specific technology / method].

[0126] In one embodiment, the anti-α4β7 antibody is its ligand (for example, mucosal adrenin, for example) For example, MAdCAM (e.g., MAdCAM1), fibronectin and / or vascular It inhibits the binding of α4β7 to 1 or more of the dressin (VCAM). Primate MAdCAM This is described in PCT Publication WO96 / 24673, and the entire teaching is revealed by reference. It is incorporated into the details. In another embodiment, the anti-α4β7 antibody inhibits the binding of VCAM. In addition, α to MAdCAM (for example, MAdCAM1) and / or fibronectin It inhibits the binding of 4β7.

[0127] In one embodiment, the anti-α4β7 antibody for use in the formulation is a mouse Act-1 antibody. It is a t-type antibody. Suitable methods for preparing humanized antibodies are well known in this art. Generally, human The anti-α4β7 antibody is a derivative of the three heavy chain complementarity determining regions (CDRs, C) of the mouse Act-1 antibody. Contains DR1, SEQ ID NO: 8, CDR2, SEQ ID NO: 9, and CDR3, SEQ ID NO: 10. It contains a heavy chain and a suitable human heavy chain framework region; and 3 of the mouse Act-1 antibody Two CDRs (CDR1, SEQ ID NO: 11, CDR2, SEQ ID NO: 12 and CDR3, distribution It will contain a light chain containing column number 13)) and a suitable human light chain framework region. Humanized Act-1 antibodies, with or without amino acid substitutions, conform to the consensus frame. It can include a suitable human framework area including a work area. For example, One or more of the framework amino acids at the corresponding positions in the mouse Act-1 antibody. It can be replaced by another amino acid, such as an acid. The constant region of the human body, or a part thereof, exists. If present, the κ or λ light chain and / or γ of human antibodies, including allele variants (even if If so, it can be derived from γ1, γ2, γ3, γ4), μ, or ε heavy chains. Specific steady-state region (For example, IgG1), its variants or some of them are selected to tailor the effector function. This can be done. For example, by incorporating a mutated constant region (mutant) into a fusion protein. This minimizes the ability to bind to Fc receptors and / or fix complement. see eg, Winter et al., GB 2,209,757 B; Morrison et al., WO 89 / 07142; Morgan et al., WO 94 / 29351, Dec. 22, 1994). The humanized form of the Act-1 antibody is PCT publication number W. As described in O98 / 06248 and WO07 / 61679, the entirety of each instruction This is incorporated herein by reference.

[0128] In another embodiment, the anti-α4β7 humanized antibody for use in the formulation is the amino acid of SEQ ID NO: 2 The heavy chain variable region including amino acids 20-140 and amino acids 20-131 of SEQ ID NO: 4 or SEQ ID NO: 5 It includes a light chain variable region containing amino acids 21-132. If desired, a suitable human constant region. For example, humanized anti-α4β7 antibody has amino acid 20 in SEQ ID NO: 2. It can contain a heavy chain containing ~470 and a light chain containing amino acids 21-239 of SEQ ID NO. 5. In another example, the humanized anti-α4β7 antibody is a heavy chain containing amino acids 20-470 of SEQ ID NO: 2. It may also contain a light chain containing amino acids 20-238 of SEQ ID NO: 4. Figure 4 shows a human antibody. This shows a sequence comparison comparing the light chain of a mouse antibody with a typical light chain. The sequence comparison reveals that the two mice Vedolizumab in which a residue is replaced with a human residue (for example, Chemical Abstract Service The humanized light chain (CAS, American Chemical Society) registration number 943609-66-3) is LDP-02 This explains that it is more human-shaped than the light chain (Figure 3). In addition, LDP-02 is somewhat hydrophobic. Alanine 114, which is highly flexible in terms of its properties, and threonine 114, which is slightly hydrophilic and contains hydroxyl molecules. And it is replaced with vedolizumab, which has 115 valine residues that are hydrophobic and potentially inward. It has a hydrophilic portion (aspartic acid 115).

[0129] Further substitutions to the antibody sequence, for example, in the framework regions of the heavy and light chains. This can be a mutation, for example, the isoleucine in residue 2 of SEQ ID NO: 14 Mutation to phosphorus; mutation of methionine to valine at residue 4 of SEQ ID NO: 14; SEQ ID NO: 1 Mutation of alanine to glycine at residue 24 of 5; mutation of alanine at residue 38 of SEQ ID NO: 15 Alanine mutation to lysine; alanine mutation to arginine at residue 40 of SEQ ID NO: 15. Mutation; mutation of methionine to isoleucine at residue 48 of SEQ ID NO: 15; SEQ ID NO: 15 Mutation of isoleucine to leucine at residue 69; at residue 71 of SEQ ID NO: 15 Mutation of arginine to valine; isoleucine mutation of threonine at residue 73 of SEQ ID NO: 15 Mutations to; or combinations thereof; CDRs of mouse Act-1 antibodies to heavy chain CDRs (CDR1, SEQ ID NO: 8, CDR2, SEQ ID NO: 9 and CDR3, SEQ ID NO: 10) Substitution by, and light chain CDRs of mouse Act-1 antibody (CDR1, sequence number) This is a substitution by (number: 11, CDR2, sequence number: 12 and CDR3, sequence number: 13). It is possible.

[0130] In some embodiments, the anti-α4β7 humanized antibody for use in the formulation is SEQ ID NO: 2 Approximately 95%, 96%, 97%, 98%, or 99% sequence identity for 20-140 amino acids. A heavy chain variable region having and amino acids 20-131 of SEQ ID NO: 4 or amino acids of SEQ ID NO: 5 It has approximately 95%, 96%, 97%, 98%, or 99% sequence identity with respect to 21-132. It includes a light chain variable region. Amino acid sequence identity is determined using initial parameters, even For example, the Lasergene method (DNASTAR, Madison, Wisconsin) This can be determined using a suitable sequence comparison algorithm. In the embodiment, used in the formulation The anti-α4β7 antibody used is vedolizumab (CAS, American Chemical Society, registration number 9436). 09-66-3).

[0131] Other α4β7 antibodies may also be used in the formulations and administration regimens described herein. The entirety of US2010 / 0254975(A) is incorporated herein by reference. The α4β7 antibody described by mgen is a formulation for treating inflammatory bowel disease in individuals. It is suitable for use in the following methods.

[0132] Anti-α4β7 antibodies can modify the nucleic acid sequences encoding each chain in living cells, such as cells in culture. It can be produced by expression. Using various host / expression vector systems The antibody molecule of the present invention can be expressed. Such a host / expression vector system is the coded This represents the medium through which the sequence is created and subsequently purified, but is an appropriate nucleotide coding sequence. Cells that have been transformed or transmigrated can express anti-α4β7 antibodies in situ. These do not include, for example, recombinant bacteria containing antibody coding sequences. Transformed with riophage DNA, plasmid DNA, or cosmid DNA expression vectors Bacteria (e.g., Escherichia coli, Bacillus subtilis); recombinant yeast expression containing antibody coding sequences Yeast transformed with vectors (e.g., Saccharomyces, Pichia) ) Microorganisms such as; recombinant virus expression vectors containing antibody coding sequences (for example For example, an insect cell line infected with a baculovirus; a set containing an antibody coding sequence. Replacement virus expression vectors (e.g., cauliflower mosaic virus, CaMV; Taba Recombinant viruses (comomosiac virus, TMV) infected with or containing antibody coding sequences Plant cell lines transformed with a plasmid expression vector (e.g., Ti plasmid); or Derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian Virus-derived (e.g., late promoter adenovirus; vaccinia virus 7). Mammalian cell lines containing recombinant expression constructs that include the 5K promoter (for example) Examples include COS, CHO, BHK, 293, 3T3, NS0), but are not limited to these. It is not possible. For example, major early and mid-stage gene promoters derived from human cytomegalovirus. It resembles a Chinese hamster ovary cell (CHO) with element-like vectors. Mammalian cells are an effective antibody expression system (Foecking et al., Gene 45:101 (1986); C ockett et al., Bio / Technology 8:2 (1990)).

[0133] In bacterial systems, numerous expression vectors are advantageously selected depending on the intended use of the antibody molecule being expressed. It is possible to produce large quantities of such proteins, for example, to generate a pharmaceutical composition of antibody molecules. If it is to be manufactured, then express a fusion protein product that is easy to purify at a high level. A vector that aims to achieve this may be desirable. Such a vector may contain a fusion protein. The antibody coding sequence is placed within the frame along with the lacZ coding region so that it can be produced. The E. coli expression vector pUR278 (Ruther et al., EMBO) can be individually linked to the vector. J. 2:1791 (1983)); pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101- Examples include 3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989). This is possible, but is not limited to, using a pGEX vector for glutathione-S-transferase. Foreign polypeptides can also be expressed as fusion proteins using ionspherase (GST). Generally, such fusion proteins are soluble, and matrix glutathione / agaro - Adsorption and binding to beads, followed by release and elution in the presence of glutathione. It can be easily purified. The pGEX vector contains the cloned target gene GST The system is designed to include a thrombin or factor Xa protease cleavage site so that it can be released from the area. It will be measured.

[0134] In insects, Autographa calif is used as a vector for expressing foreign genes. Ornica nuclear polyhedron disease virus (AcNPV) is used. The virus is Spodop It proliferates within Tera frugiperda cells. The antibody coding sequence is individually... Cloning into non-essential regions of viruses (e.g., polyhedrin genes) and AcNPV It is placed under the control of a motor (for example, a polyhedrin motor).

[0135] In mammalian host cells, numerous virus-based expression systems can be used. Adenovirus When used as an expression vector, the antibody coding sequence is used in relation to adenovirus It can be linked to transcription / translation regulatory complexes, such as late promoter and segmental leader sequences. Next, this chimeric gene is converted into an adenovirus by recombination in vitro or in vivo. It can be inserted into the genome. In non-essential regions of the viral genome (for example, region E1 or E3) The inserted recombinant virus is capable of surviving in the infected host and expressing antibody molecules. This can cause problems (for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (19 See 84). Efficient translation of the inserted antibody coding sequence requires a specific start signal. These may also be required. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon synchronizes with the reading frame of the desired coding sequence and inserts the entire sequence. Body translation must be ensured. These exogenous translation control signals and start codons It may be of various origins, and can be both natural and synthetic. The efficiency of expression depends on the appropriate transcription. This can be enhanced by including enhancer elements, transcription termination factors, etc. (Bittner et al.) See I., Methods in Enzymol. 153:51-544 (1987).

[0136] In addition, it regulates the expression of the inserted sequence and modifies the gene product in a desired specific manner. The host cell line to be reduced can be selected. Such modifications of protein products (e.g.) Protein function is important for the function of proteins (e.g., glycosylation) and processing (e.g., cleavage). It may be necessary. Various host cells perform post-translational processing of proteins and gene products and The modification has distinctive characteristics and specific mechanisms. Select an appropriate cell line or host system. To ensure the correct modification and processing of expressed foreign proteins, Regarding the proper processing of subsequent transcripts, glycosylation and phosphorylation of gene products in cells Eukaryotic host cells with a mechanism can be used. Such mammalian host cells include Chinese Hamster ovaries (CHO), NS0, HeLa, VERY, young hamster kidneys (BHK) ), monkey kidney (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma cells ( For example, HepG2), for instance, BT483, Hs578T, HTB2, BT20 and Breast cancer cell lines such as T47D, as well as, for example, CRL7030 and Hs578Bst Examples of normal mammary gland cell lines include, but are not limited to, those listed above.

[0137] The glycosylation mechanisms of various cell types involve antibodies with different glycosylation compositions compared to other cell types. It is possible to produce antibodies that are not glycosylated, similar to bacteria. In one embodiment, the cell type for producing anti-α4β7 antibody is NS0 cells or CHO cells. These are mammalian cells. In one aspect, mammalian cells are involved in cellular mechanisms. This may include enzyme deletions, and such exogenous genes may be involved in, for example, transformation or transduction. Therefore, a construct or vector for introduction into cells is used to operably link the replacement enzyme. It is possible. A construct or vector containing an exogenous gene may invite the construct or vector. Selectively stimulates cells to produce polypeptides encoded by constructs or vectors. To confer a specific location. In one embodiment, CHO cells are deleting the dihydrofolate reductase gene or These are DG44 cells including inactivation (Chasin and Urlaub (1980) PNAS USA 77:4216). In another embodiment, CHO cells include C with a deletion or inactivation of the glutamine synthase enzyme. HOK1 cells (for example, U.S. Patent No. 5,122,464 or No. 5,827) (See issue 739).

[0138] Solid Dosage Form The solid formulations of the present invention are generally prepared by drying liquid formulations. Drying methods, such as freeze-drying or spray-drying, can be used. Containers that would normally be used to store, ship, and distribute pharmaceutical products (for example) This involves drying the liquid formulation in a vial (for example, Gatlin and Nail in Protein Purification Process Engineering, ed. Roger G. Harrison, Marcel Dekker See Inc., 317-367 (1994). Once the formulation is frozen, the atmospheric pressure is lowered, and the temperature For example, the process is adjusted to remove the freezing solvent via sublimation. This stage is sometimes called primary drying. If desired, the temperature can then be raised to dry the preparation. Naturally, the binding solvent can be removed by evaporation. This step in the freeze-drying process is two This is sometimes called secondary drying. When the formulation reaches the desired degree of dryness, the drying process is terminated, and the container is opened. Seal the container. The final solid formulation is sometimes called a "freeze-dried formulation" or "cake." The freeze-drying process can be carried out using suitable equipment. There are many suitable freeze-drying devices. Available from commercial sources (for example, SP in Stone Ridge, New York). (Scientific).

[0139] Using various suitable apparatuses, liquid formulations are dried to produce solid (e.g., lyophilized) formulations. It can be manufactured. Generally, lyophilized formulations are made from vials of liquid formulations that are dried on top of them. Prepared by those skilled in the art using a sealed chamber containing a shelf on which the material is placed. Shelf temperature This can be controlled, like the cooling rate, heating rate, and the pressure inside the chamber. The various process parameters discussed in the specification refer to the processes carried out using this type of apparatus. It will be understood that a person skilled in the art can, if desired, use the parameters described herein. It can be easily adapted to other types of drying equipment.

[0140] The suitable temperature and vacuum level for primary and secondary drying can be easily determined by those skilled in the art. It is possible. Generally, formulations are frozen at temperatures below approximately -30°C, for example, -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 approximately 10°C, 20°C, 30°C, 40°C, or 50°C above the freezing point. It is carried out at a warm temperature. In one embodiment, the primary drying conditions are the glass transition temperature of the formulation. Alternatively, the anti-α4β7 antibody can be maintained below the decay temperature. Necrophilia When the temperature exceeds a certain level, the amorphous frozen matrix flows (breaks down), and the protein molecules become rigid solid. The protein molecule may not be surrounded by trix, and may not be stable in the breakdown matrix. This could result in a situation where the formulation does not dry completely. Furthermore, if disintegration occurs, the formulation may not dry completely. There is a result in a high water content in the formulation, which can lead to a high rate of protein degradation. The amount of time that freeze-dried products can be stored before their quality deteriorates to an unacceptable level may decrease. In one embodiment, the shelf temperature and chamber pressure are kept below the decay temperature during the primary drying process. The glass transition temperature of the cryopreservation agent is selected to maintain the product. It can be measured by method, for example, differential scanning calorimetry (DSC). Temperature is determined by methods known in the art, for example, freeze-drying microscope, optical coherence It can be measured by tomography. The ratio of non-reducing sugars to proteins (moles:moles) and The amounts of other formulation components will affect the glass transition temperature and decay temperature in some embodiments. The glass transition temperature of the α4β7 antibody preparation is approximately -35°C to approximately -10°C, and approximately -35°C to approximately - The temperature is 25°C, or approximately -35°C to approximately -29°C. In another embodiment, the α4β7 antibody preparation is gas The glass transition temperature is approximately -29°C. In some embodiments, the glass transition temperature of the α4β7 antibody preparation is The temperature ranges are approximately -30°C, -31°C, -32°C, -33°C, -34°C, and -35°C. Or it is about -36°C. In some embodiments, the decay temperature of the α4β7 antibody preparation is about -30°C. The ranges are approximately 0°C to 0°C, approximately -28°C to approximately -25°C, or approximately -20°C to approximately -10°C. In this state, the decay temperature of α4β7 antibody preparations is approximately -26°C. (This is not bound by a specific theory.) Undesired, the faster the rising speed, the higher the product's collapse temperature. Primary drying process This involves removing at least 50%, at least 60%, or at least 70% or more of the solvent. This can be done. In one embodiment, the primary drying step removes more than 80% of the solution from the anti-α4β7 antibody preparation. Mediator.

[0141] Primary drying depends on the temperature and pressure of the rack. The conditions for primary drying are different process parameters. It can be empirically determined by freeze-drying at the source. Primary drying is also based on the product temperature. Therefore, it can be mathematically modeled. The equations for mass and thermal transition, combined with knowledge of Rp and Kν, ( According to Milton, et al. (1997) PDA J of Pharm Sci & Tech, 51: 7-16), shelf temperature and A combination of input variables, including process input variables such as formulation variables captured by pressure and Rp value. This makes it possible to understand decay and interaction. These models involve decay temperature and instrument capacity. This helps determine the parameters used in efficient processes based on the product temperature limits. Tsu.

number

number

[0142] Equation 1 is given by container (A p Sublimation rate (dm / dt) during primary drying with respect to the inner cross-sectional area of ​​) , ice vapor pressure (P0), chamber pressure (P c ) and aspects of the cake and stopper The mass transition resistance (R) of the product is normalized. pRegarding this, P0 at the sublimation interface is determined from equation 2. It can be determined that P0 is related to the temperature of the product ice at the sublimation interface, and it is the product temperature (T p This is an approximation from, and can be measured by a thermocouple at the bottom of the vial, or other The above equation can be derived from the determination of the variables. Equation 3 is derived from the vial from the shelf. It is related to the thermal transition rate to A ν is the area of ​​the vial, and K ν The thermal transition coefficient of the vial Yes, T s This is the temperature of the shelf, T p is the product temperature. Equation 4 is the rate of heat-mass transfer. Combine the equations, ΔH s This is the heat of sublimation.

[0143] As can be seen from the equation for primary drying, the shelf temperature (T s ), product temperature (Tp), Chamber pressure (P c ), the mass transition resistance of the cake (R p ) and thermal transition coefficient (K ν ) is rising It can affect the speed of the flower.

[0144] Any step after freezing and before primary drying is annealing. In this step, a freeze dryer is used. The shelf temperature is adjusted for a short period of time, for example, about 2-6 hours, about 3-5 hours, or about 4 hours, to allow the formulation to dissolve. The temperature is raised above the glass transition temperature, and then the shelf temperature is lowered again below the glass transition temperature of the formulation. Annealing is used to crystallize the filler and form larger, more uniform ice crystals. The annealing process results in a cake that is dried without annealing. A dried cake has a larger surface area, which can affect the reconstitution time. The annealing process for α4β7 antibody preparations is approximately -30°C to approximately -10°C, or approximately -25°C to approximately The temperature can be -15°C. In one embodiment, annealing for α4β7 antibody preparations The temperature is approximately -20°C.

[0145] Secondary drying is generally carried out at a temperature above the freezing point of the liquid formulation. For example, secondary drying This can be carried out at approximately 10°C, 20°C, 30°C, 40°C, or 50°C. In one method, the secondary drying temperature is room temperature, for example, 20-30°C. The duration of the secondary drying is It should be sufficient for the moisture content to be reduced to <5%.

[0146] In another embodiment, the freeze-drying cycle includes freezing at approximately -45°C and annealing at approximately -20°C. Ring, refreezing at approximately -45°C, and primary drying at approximately -24°C and 150 millitol, This includes secondary drying at approximately 27°C and 150 millitol.

[0147] R p This is due to the solid content of the frozen DP and the thermal history of the DP which affects the pore structure of the cake. The process is affected by the stages of freezing, annealing, and refreezing. The thermal history is also affected by the secondary drying stage. It can also affect the floor, and a larger surface area can help with water desorption (Pika l, et al. (1990) Int. J. Pharm., 60: 203-217). Between the primary and secondary freeze-drying stages. Useful process parameters to control include shelf temperature and chamber temperature between each stage of the drying cycle. It could be a form of pressure.

[0148] To scale up the process, the load on the freeze dryer and the solid content can affect the drying cycle. The time required for primary drying can be affected by the solid content of the formulation. At high solid content, for example, For example, is the concentration of the entire solid (excipients and / or proteins) the formulation that determines the drying time? These exceed 10 w / v%, or exceed 15 w / v%, for example, 50-100% When variations occur, drying time can be an effect. For example, formulations with a high solid content may have a low solid content. It may have a longer drying time than the formulation with a higher content. In some embodiments, the capacity of the freeze dryer is utilized. The rate can range from approximately 25% to approximately 100%. At high capacity load percentages, low capacity The primary drying time can increase up to twice as much as the load percentage. As the solid content increases, different The difference between primary drying times at different load percentages increases. In one embodiment, the solid content is 20-25 It is less than %, and the load is between 25% and 100%.

[0149] The vial size is selected based on the surface area exposed to the shelf and vacuum during freeze-drying. It is possible. Since the drying time is directly proportional to the height of the cake, the size of the vial is reasonable. It can be selected based on what is determined to be the height of the cake. Vials with a diameter have large contact with the shelf for efficient heat transfer during the freeze-drying cycle. This can provide a solution. Diluted antibody solutions with a large liquid volume require more time to dry. This will be essential. Larger vials are more expensive to store and transport, and require more upper space and manufacturing space. It has a high ratio of the agent, and the formulation with a high ratio can be exposed to the decomposition effect of moisture during long-term storage, It is necessary to balance the vial size with the volume of the formulation. For the 300 mg dose... The anti-α4β7 antibody preparation is available in 3ml, 5ml, 6ml, 10ml, and 20ml sizes before lyophilization. It can have a volume of 50 ml or 100 ml. In one embodiment, the vial is Iz is 20 ml of a 60 mg / ml solution for a 300 mg dose.

[0150] After freeze-drying, the vials are sealed under vacuum, for example, by being sealed with a stopper. Alternatively, Prior to sealing, a gas, such as dry air or nitrogen, may be placed inside the container. If oxidation is a concern, gas can be introduced into the freeze-drying chamber, and the acidity of the freeze-dried product can be reduced. It may include a gas that slows down or hinders the transformation. In one embodiment, the gas is not oxygen-containing. Gases, for example, nitrogen or inert gases, for example, helium, neon, argon, crypt In another embodiment, the gas is nitrogen or argon.

[0151] In some embodiments, the volume of the anti-α4β7 antibody preparation before lyophilization is reconstituted before administration. It is the same volume as the solution. For example, a preparation that is approximately 5.5 ml before freezing will have the same volume as a dry solid. By adding a volume of liquid to consider the product, such as water or saline solution, approximately 5.5 ml It can be reconstituted to a volume different from that of the solution being reconstituted. In other embodiments, the volume of the solution being reconstituted is different from that of the solution being reconstituted. It is desirable to freeze-dry the anti-α4β7 antibody preparation in a certain volume. For example, anti-α4β7 antibody The formulation is freeze-dried as, for example, a 0.25×, 0.5×, or 0.75× diluted solution. Reconstruction to 1× by adding 75%, half, or 25% less liquid than before freeze-drying. This can be achieved. In this embodiment, a 300 mg dose is diluted with 5% sucrose to 30 mg / m². The antibody solution is freeze-dried and reconstituted into a 60 mg / ml antibody solution with 10% sucrose. It is possible to further modify the freeze-dried anti-α4β7 antibody preparation compared to the preparation before freezing. It can be reconstituted into a diluted solution.

[0152] Treatment with antibody preparations In one embodiment, the present invention is effective, for example, in treating a disease or disorder in humans. This includes administering the anti-α4β7 antibody preparation described herein in a specified amount to the target. To provide a method for treating a disease or disorder. Human subjects are adults (e.g., 18 years of age or older). The subjects may be young people or children. The human subjects may be people aged 65 years or older. Alternative therapeutic dosing device In contrast to the illustration, human subjects aged 65 years or older require modifications to the dosing regimen described herein. Instead, conventional anti-α4β7 antibody preparations described herein may be administered.

[0153] The target group is those lacking an appropriate response to immunomodulatory factor TNF-α antagonists or combinations thereof. They either lacked a response to it or were unwilling to accept treatment based on it. It is possible. The patient has received at least one corticosteroid for inflammatory bowel disease. For example, they may have previously received treatment with prednisolone. An inappropriate response to iodine may be treated with prednisolone 3 daily orally for 2 weeks or intravenously for 1 week. Despite experience with at least one 4-week induction plan including a dose equivalent to 0 mg, persistence It refers to the signs and symptoms of an active disease. A lack of response to corticosteroids every Gradually reduce the dose of corticosteroids to a level equivalent to or lower than 10 mg of oral prednisolone per day. This refers to two failed attempts. Corticosteroid intolerance is associated with Cushing's syndrome. Pre-existing conditions include osteopenia / osteoporosis, hyperglycemia, insomnia, and / or a history of infection.

[0154] Immunomodulators include, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. It may be xate. An inappropriate response to immunomodulatory agents is at least one oral bruise. Thiopurine (≧1.5 mg / kg), 6-mercaptopurine (≧0.75 g / kg) or Despite experience with an 8-week plan of methotrexate (≥12.5g / kg), the symptoms persisted. This refers to signs and symptoms of an active disease. Immunomodulatory intolerances include vomiting / nausea, abdominal pain, Pancreatitis, LFT abnormalities, lymphopenia, TPMT gene mutations, and / or infections are among the possible causes. However, it is not limited to these.

[0155] In one instance, the patient lacked an adequate response to TNF-α antagonist therapy, There may have been a lack of response to this, or a lack of acceptance of it. NF-α antagonists are, for example, those that inhibit the biological activity of TNF-α, preferably TNF-α A binding agent, for example, a monoclonal antibody, for example, REMICADE (inflicting Ximab, HUMIRA (adalimumab), CIMZIA (certolizumab pecol) , such as SIMPONI (golimumab) or ENBREL (etanercept) It is a circulating receptor fusion protein. An inappropriate response to TNF-α antagonists is caused by inflammation. Two doses of ximab 5 mg / kg IV at least two weeks apart; one dose of 80 mg adalimumab. One subcutaneous dose, followed by a single 40 mg dose at least two weeks apart; or a 400 mg cell dose. Subcutaneous administration of torizumab pecol, at least two doses at least once every four weeks, spaced at least two weeks apart. TNF refers to persistent signs and symptoms of disease despite the experience of inter-induction therapy. - Lack of response to α-antagonists indicates recurrence of symptoms between maintenance doses following the previous clinical benefit. This refers to the intolerance of TNF-α antagonists, including infusion-related reactions, demyelination, congestive heart failure and / or Infection is one example, but it is not limited to these.

[0156] Loss of maintenance of remission is used herein in reference to patients with ulcerative colitis, Mayo This refers to an increase of at least 3 points in the score and at least 2 points in the modified Baron score.

[0157] In another aspect, the present invention relates to (1) the development of α4β7 integrin in vitro and / or in vivo. (2) the activity or function of α4β7 integrin, for example, (a) binding Combined functions (for example, MAdCAM of α4β7 integrin (for example, MAdCAM1)) (b) Ability to bind to fibronectin and / or VCAM-1 in tissue Leukocyte infiltration, including the recruitment and / or accumulation of leukocytes (e.g., phosphorus into intestinal mucosal tissue). The present invention provides an anti-α4β7 antibody preparation that can regulate the ability to inhibit the migration of Pacyl cells. In this embodiment, the antibody in the formulation can conjugate α4β7 integrin, and its liga (For example, MAdCAM (for example, MAdCAM1), VCAM-1, Fibro) It is possible to inhibit the binding of α4β7 integrin to one or more nectin molecules, thereby This inhibits the infiltration of leukocytes into tissues (including the recruitment and / or accumulation of leukocytes in tissues). In another embodiment, the antibody in the formulation can conjugate α4β7 integrin, Ligands (e.g., MAdCAM (e.g., MAdCAM1), VCAM-1, F By selectively inhibiting the binding of α4β7 integrin to 1 or more of ibronectin, This leads to the infiltration of leukocytes into tissues (including the recruitment and / or accumulation of leukocytes in tissues). It inhibits (m). Such anti-α4β7 antibody preparations are used in vitro and / or in vivo in the gastrointestinal tract. Includes related tissues, lymphatic organs, or white blood cells (especially lymphocytes such as T cells or B cells). In mucosal tissue, it inhibits cell adhesion of cells containing α4β7 integrin to vascular endothelial cells. This can be done. In yet another embodiment, the anti-α4β7 antibody preparation of the present invention is an MA of α4β7. Inhibits interaction with dCAM (e.g., MAdCAM1) and / or fibronectin. It is possible. Furthermore, in yet another embodiment, the anti-α4β7 antibody preparation of the present invention is For example, selectively inhibiting the interaction of α4β7 with VCAM and MAd Inhibits interaction with CAM (e.g., MAdCAM1) and / or fibronectin. It is possible.

[0158] Using the anti-α4β7 antibody preparation of the present invention, the binding function of α4β7 integrin and / or white It regulates (e.g., inhibits) the infiltration function of blood cells (e.g., lymphocytes, monocytes). It can (or prevent) the formation of white blood cells (e.g., lymphocytes, monocytes) in tissue. In particular, infiltration into tissues expressing the molecule MAdCAM (for example, MAdCAM1) (tissue In accordance with the methods used in the treatment of diseases related to the recruitment and / or accumulation of white blood cells in the body. This inhibits the binding of α4β7 integrin to ligands (i.e., one or more ligands). Humanized immunoglobulin can be administered.

[0159] To treat such diseases, an effective amount of the anti-α4β7 antibody preparation of the present invention is administered to an individual (for example) For example, it is administered to mammals such as humans or other primates. (including endothelial cells of the stratum), other mucosal tissue or the molecule MAdCAM (e.g., MAdCAM1) Tissues that express (for example, gastrointestinal tissues such as the small veins of the lamina propria of the small and large intestines) This method treats inflammatory diseases, including those related to tissues and mammary glands (e.g., breastfeeding mammary glands). Treatment can be performed in accordance with the law. Similarly, MAdCAM (e.g., MAdCAM1) Diseases associated with the infiltration of leukocytes into tissues as a result of leukocyte binding to cells expressing a specific gene. Individuals suffering from the disease can be treated according to the present invention.

[0160] In one embodiment, diseases that can thus be treated include, for example, ulcerative colitis, Intestinal diseases associated with Rohn's disease, ileitis, celiac disease, non-tropical sprue, and seronegative arthropathy. It occurs in patients with microscopic or collagenous colitis, neutrophilic gastroenteritis, or after rectal and colectomy. Examples include inflammatory bowel disease (IBD) such as pouchitis and ileoanal anastomosis. Preferably Inflammatory bowel disease is Crohn's disease or ulcerative colitis. Ulcerative colitis is moderate to severe. It could be severe active ulcerative colitis. Treatment is for moderate to severe active ulcerative colitis. Mucosal healing may occur in suffering patients. Treatment also involves the patient administering corticosteroids. This may result in a reduction or elimination of use, or both.

[0161] Pancreatitis and insulin-dependent diabetes can be treated with the formulation of the present invention. It is a disease. MAdCAM (for example, MAdCAM1) is BALB / c and SJL Similar to Uss, some blood vessels in the exocrine glands of the pancreas originate from NOD (non-obese diabetes) It has been reported that MAdCAM is expressed in NOD mice. MAdCAM is induced on endothelial cells in inflamed pancreatic islets, and in the early stages of pancreatic insular inflammation. At that stage, it was the dominant adresin expressed by the islet endothelium of NOD (Hanninen, A., et al.). al., J. Clin. Invest., 92: 2509-2515 (1993)). Anti-MAdCAM antibody or anti-α4β7 Treatment of NOD mice with antibodies prevented the development of diabetes (Yang et al., Diabetes, 46: 1542-1547 (1997). Furthermore, accumulation of lymphocytes expressing α4β7 was observed within the island. MAdCAM-1 is released to the blood vessels of inflamed islets (Hanninen, A., et al., J. Clin. Inv est., 92: 2509-2515 (1993)) or gastrointestinal lesions in mantle cell lymphoma (Geissman n et al., Am. J. Pathol., 153:1701-1705 (1998)) Lymphoma cell clusters mediated by α4β7 He was deemed to be involved in the agreement.

[0162] Examples of inflammatory diseases related to mucosal tissue that can be treated with the formulation of the present invention include: Cholecystitis, cholangitis (Adams and Eksteen Nature Reviews 6:244-251 (2006) Grant et al., Hepatology 33:1065-1072 (2001), for example, primary sclerosing cholangitis, for example, intestinal vesicles - Chet's disease, or pericholangitis (tissue surrounding the bile ducts and liver), and graft-versus-host disease ( For example, in the gastrointestinal tract (for instance, after bone marrow transplantation) (Petrovic et al. Blood 103:1542-1547 ( One example is 2004). As seen in Crohn's disease, inflammation can spread beyond the mucosal surface. Because there are many such conditions, for example, sarcoidosis, chronic gastritis, for example, autoimmune gastritis (Ka Takai et al., Int. Immunol., 14:167-175 (2002)) and other chronic conditions such as idiopathic conditions. These inflammatory diseases are treatable.

[0163] The present invention relates to a method for inhibiting the infiltration of leukocytes into mucosal tissue. The present invention also relates to cancer (for example) For example, this also relates to methods for treating α4β7-positive tumors such as lymphoma. Other examples of inflammatory diseases involving mucosal tissue that can be treated with this include mastitis (mammary gland ) and irritable bowel syndrome are examples.

[0164] The etiology of this disease utilizes the interaction between α4β7 and MAdCAM (e.g., MAdCAM-1). The disease or pathogen to which the preparation is used is curable by the anti-α4β7 antibody in the preparation described herein. It can be treated. Examples of such diseases include, for example, human immunodeficiency virus (even For example, immunodeficiency disorders caused by (see WO2008 / 140602) can be cited.

[0165] The formulation of the present invention is administered in an amount effective in inhibiting α4β7 from binding to its ligand. Regarding treatment methods, the effective amount is needed to achieve the desired therapeutic effect (including prevention). This should be sufficient (for example, binding and / or signal transduction mediated by α4β7 integrin). This reduces or inhibits leukocyte adhesion and infiltration and / or related cellular responses. (An amount sufficient to inhibit it). An effective amount, for example, saturating α4β7 integrin, even Therefore, anti-α4β7 antibodies with sufficient titer to maintain neutralization are clinically important in inflammatory bowel disease. It can induce a response or remission. The formulation of the present invention is administered in a single dose or multiple doses. It is possible. The dosage can be determined by methods known in the art, even However, this may depend on the individual's age, sensitivity, tolerance, and overall health. Examples of administration methods include For example, local routes such as intranasal, inhalation, or transdermal administration, for example, supply tubes. or enteral routes via suppositories, and for example, intravenous, intramuscular, subcutaneous, intra-arterial, intraperitoneal or nitrate. Parenteral routes, such as intracellular administration, are one example. The appropriate dosage of antibody is approximately 0 per treatment. 0.1 mg / kg body weight to approximately 10.0 mg / kg body weight, for example, approximately 2 mg / kg to approximately 7 mg The concentration may be approximately 3 mg / kg to 6 mg / kg, or approximately 3.5 to 5 mg / kg. In certain embodiments, the dose administered is approximately 0.3 mg / kg, approximately 0.5 mg / kg, approximately 1mg / kg, approx. 2mg / kg, approx. 3mg / kg, approx. 4mg / kg, approx. 5mg / kg, approx. 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, or approximately 10 mg / kg It is g.

[0166] For example, dilution of a recomposed anti-α4β7 antibody (e.g., physiological saline or 5%) The final dosage form after dextrose infusion is approximately 0.5 mg / ml for administration. The dosage can be approximately 5 mg / ml. The final dosage form is 1.0 mg / ml to approximately 1.4 mg. / ml, about 1.0mg / ml~about 1.3mg / ml, about 1.0mg / ml~about 1.2mg / ml, about 1.0~1.1mg / ml, about 1.1mg / ml~1.4mg / ml, approx. 1.1mg / ml ~ approx. 1.3mg / ml, approx. 1.1mg / ml ~ approx. 1.2mg / ml, approx. 1.2 mg / ml to approximately 1.4 mg / ml, approximately 1.2 mg / ml to approximately 1.3 mg / ml or The concentration may be between approximately 1.3 mg / ml and approximately 1.4 mg / ml. The final dosage form is approximately 0 .6mg / ml, 0.8mg / ml, 1.0mg / ml, 1.1mg / ml, approx. 1.2m g / ml, approx. 1.3mg / ml, approx. 1.4mg / ml, approx. 1.5mg / ml, approx. 1.6m The concentration may be g / ml, approximately 1.8 mg / ml, or approximately 2.0 mg / ml. In one embodiment... In one embodiment, the total dose is 180 mg. In another embodiment, the total dose is 300 mg. For administration of a dose of mg of anti-α4β7 antibody, use 250 ml of physiological saline or 5% dextrose. It can be diluted with a rose solution.

[0167] In some embodiments, the administration plan has two phases: an induction phase and a maintenance phase. In the induction phase, for example, For example, inducing immune tolerance to an antibody or its antigen-binding fragment, or inducing a clinical response. An effective amount suitable for specific purposes such as improving the symptoms of inflammatory bowel disease. A method that rapidly provides an antibody or its antigen-binding fragment, It is administered. If the patient is being treated with anti-α4β7 antibody for the first time, the anti-α4β7 antibody treatment Since the law was enacted, for example, more than 3 months, more than 4 months, more than 6 months, more than 9 months If you have not received treatment for a long period of time, more than one year, more than 18 months, or more than two years, During the maintenance phase of anti-α4β7 antibody therapy, there is a recurrence of symptoms of inflammatory bowel disease, for example, disease remission. If there is a recurrence, induction phase treatment can be administered. In some embodiments, induction phase The plan is to maintain an average trough serum concentration higher than the average steady-state trough serum concentration maintained between maintenance plans. The serum concentration, for example, the concentration immediately preceding the next dose, is produced.

[0168] In the maintenance phase, the antibody or its antigen-binding fragment is the stable antibody or its antigen-binding fragment achieved in induction therapy. It is administered in a manner that maintains the response at the level of the antigen-binding fragment. The maintenance plan is symptomatic. It can prevent recurrence of the disease or inflammatory bowel disease. The maintenance plan is, for example, simple injections. To provide convenience to patients who do not frequently visit outpatient clinics for planned treatment or therapy. This is possible. In some embodiments, the maintenance plan may include low dose, small doses, self-administration and pre-administration. A strategy selected from a group consisting of the combinations described herein, for example, This includes administration of an anti-α4β7 antibody or its antigen-binding fragment contained in the formulation.

[0169] In one embodiment, for example, during the induction phase of the treatment, the administration plan is to address inflammation in a human patient. An effective amount of anti-α4β in the formulations described herein for inducing remission of genital colon disease The present invention provides an antibody or antigen-binding fragment. In some embodiments, an effective amount of anti-α4β7 antibody is provided. By the end of the induction phase, the concentration was approximately 5 μg / ml to 60 μg / ml, and approximately 15 μg / ml to 45 μg / ml. / ml, approximately 20μg / ml to approximately 30μg / ml, or approximately 25μg / ml to approximately 35μg / ml This is sufficient to achieve the mean trough serum concentration of 1 anti-α4β7 antibody. Duration of the induction phase The treatment may last approximately 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In embodiments, the induction plan is, for example, an anti-α4β7 anti-formulation in the formulations described herein. High doses, frequent administration of the body or its antigen-binding fragments, and combinations of high doses and frequent administration. A strategy can be used to select from a group consisting of [number] doses. Induction administration may be one or more than one dose. Multiple doses may be administered, for example, at least two doses. During the induction phase, the dose is once daily. Administer every other day, twice a week, once a week, once every 10 days, once every two weeks, or once every three weeks. In some embodiments, the induction dose is administered within the first two weeks of treatment to prevent α4β 7. It is administered by antibody. In one embodiment, induction is administered once at the start of treatment (day 0). And it may be once, about two weeks after the start of treatment. In another embodiment, the duration of the induction phase is 6 It is a week. In another embodiment, the duration of the induction phase is 6 weeks, with multiple in the first two weeks. An induction dose is administered.

[0170] In some embodiments, for example, patients with severe inflammatory bowel disease (e.g., anti-TNF- When initiating treatment for patients who have not responded well to alpha therapy, the induction phase is mild or moderate. It needs to have a longer duration than the patient. In some embodiments, the induction phase of severely ill patients This includes at least 6 weeks, at least 8 weeks, at least 10 weeks, and at least 12 weeks. Or it can have a duration of at least 14 weeks. In one embodiment, severe disease The induction dosing plan for the patient involves administration at week 0 (initiation of treatment), administration at week 2, and administration at week 6. This may include the administration of [a drug]. In another embodiment, the induction dosing plan for a patient with a severe illness is This includes administration at week 0 (start of treatment), week 2, week 6, and week 10. It is possible.

[0171] In one embodiment, for example, during the maintenance phase of the treatment, the administration plan is to measure mean steady-state trough blood Concentration levels, for example, approximately 5 to 25 μg / mL, approximately 7 to 20 μg / mL, approximately 5 to 10 μg / mL g / mL, approximately 10-20 μg / mL, approximately 15-25 μg / mL, or approximately 9-13 μg Maintain a plateau concentration of anti-α4β7 antibody at / mL immediately before the next dose. In another embodiment, The dosage plan is, for example, during the maintenance phase of treatment, approximately 20-30 μg / mL, approximately 20-5 5 μg / mL, approximately 30-45 μg / mL, approximately 45-55 μg / mL, or approximately 35-4 Maintain an average steady-state trough serum concentration of 0 μg / mL of anti-α4β7 antibody.

[0172] Dosage: Once per week, once every two weeks, once every three weeks, once every four weeks, every six weeks It can be administered once every 8 weeks, or once every 10 weeks. This refers to frequent doses, for example, once a week, once every two weeks, once every three weeks, every four weeks. Once per month, to induce remission of an active disease or to treat a new patient, even Therefore, it may be useful in inducing tolerance to anti-α4β7 antibodies. Infrequent doses, For example, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, or 1 A dose of once every week is used for prophylactic therapy, for example, to maintain remission in patients with chronic diseases. This may be useful for that purpose. In one embodiment, the treatment plan is approximately day 0, week 2, and week 6. and thereafter treatment every 4 or 8 weeks. In one embodiment, the maintenance medication plan includes every 8 weeks This includes administration. Patients receiving a maintenance medication regimen of once every 8 weeks have one or more diseases. In embodiments where the patient experiences a recurrence of symptoms, for example, a relapse, the number of doses may be, for example, 4. It can be increased to once a week.

[0173] The medication is administered to the patient over a period of approximately 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes. It is possible.

[0174] The administration plan is designed to induce clinical response and clinical remission in patients with inflammatory bowel disease. It can be optimized for the brain of the patient being treated. In some embodiments, the administration plan is optimized for the brain of the patient being treated. The ratio of CD4 to CD8 in the cerebrospinal fluid remains unchanged.

[0175] In some cases, prolonged clinical remission, for example, within 6 months or 1 year after the start of treatment, may occur. This persists through at least two, at least three, and at least four outpatient visits with the attending physician. Clinical remission can be achieved through an optimized dosing regimen.

[0176] In some cases, a prolonged clinical response, for example, lasting at least six months after the start of treatment, A clinical response lasting at least 9 months and at least 1 year is achieved with an optimized dosing regimen. It can be achieved.

[0177] In one embodiment, the administration plan is as follows: an initial dose of 300 mg, and approximately two weeks after the initial dose, 300 mg A second follow-up dose of mg, a third follow-up dose of 300 mg approximately 6 weeks after the initial dose, and thereafter, This includes a fourth subsequent dose administered every 4 weeks or every 8 weeks following the third subsequent dose.

[0178] In some embodiments, the method, dosage, or administration plan of treatment is such that the patient is resistant to anti-α4β7 antibodies. It reduces the likelihood of the HAHA reaction occurring, for example, when reactive to anti-α4β7 antibodies. The occurrence of HAHA, as measured by antibodies, increases the clearance of anti-α4β7 antibodies. It is possible to lower the serum concentration of anti-α4β7 antibody, for example, α It reduces the number of anti-α4β7 antibodies bound to 4β7 integrin, thus lowering the therapeutic effect. In some embodiments, to prevent HAHA, a maintenance plan is followed by an induction plan. This allows us to treat patients. In some embodiments, there is a rest period between the induction plan and the maintenance plan. There is no waiting period. In some embodiments, the induction plan involves administering multiple doses of anti-α4β7 antibody to the patient. This includes initiating treatment with anti-α4β7 antibodies to prevent HAHA. High initial dose, for example, at least 1.5 mg / kg, at least 2 mg / kg, low At least 2.5 mg / kg, at least 3 mg / kg, at least 5 mg / kg, and at least Also 8 mg / kg, at least 10 mg / kg or about 2 to about 6 mg / kg, or frequent initial doses Dosage, for example, approximately once a week, approximately once every two weeks, or approximately once every three weeks Patients can be treated with a standard dose. In some embodiments, the treatment method is used for a small number of patients. 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% are considered HAHA negative. To maintain. In other embodiments, the treatment method is for at least 6 weeks, at least 10 weeks, At least 15 weeks, at least 6 months, at least 1 year, at least 2 years, or recovery The duration of the therapy is to maintain the patient as HAHA-negative. In some embodiments, the patient Or at least 30%, at least 40%, and at least 5% of patients who have developed HAHA. 0% or at least 60% maintain a low titer of anti-α4β7 antibody, for example, ≤125. In one embodiment, the treatment method involves treating at least 70% of patients with an anti-α4β7 antibody. Maintain a HAHA-negative status for at least 12 weeks after initiation.

[0179] The formulation can be administered to an individual (e.g., a human) alone or in combination with another agent. The formulation can be administered before, together with, or after the administration of additional agents. Morphologically, there is more than one formulation that inhibits the binding of α4β7 integrin to its ligand. In such embodiments, an agent, for example, anti-MAdCAM or anti-VCAM-1, is administered. Monoclonal antibodies, such as monoclonal antibodies, can be administered. Another embodiment Therefore, the additional agent inhibits the binding of leukocytes to endothelial ligands via a pathway different from the α4β7 pathway. It inhibits the expression of chemokine (CC motif) receptor 9 (CCR9). Lymphocytes express chemokines (TECK or CCL25) or LFA-1 in the thymus. It can inhibit the binding of agents that inhibit binding to intracellular adhesion molecules (ICAMs). For example, anti-TECK antibody or anti-CCR9 antibody or PCT publication WO03 / 099773 or Small molecule CCR9 inhibitors, such as those disclosed in WO04 / 046092, or anti-IC In addition to the formulation of the present invention, an AM-1 antibody or an oligonucleotide that inhibits ICAM expression is added to the formulation of the present invention. In yet another embodiment, an additional active ingredient (for example, sulfur) is administered. Asalazine, azathioprine, 6-mercaptopurine, 5- Aminosalicylic acid-containing anti-inflammatory Anti-inflammatory compounds such as drugs, other non-steroidal anti-inflammatory compounds, steroidal anti-inflammatory compounds Compounds, or antibiotics commonly administered for the control of IBD (e.g., ciprofloxa) The present invention uses syn, metronidazole, or other biological agents (e.g., TNFα antagonists) It can be administered in combination with other formulations.

[0180] In the embodiment, the dose of the drug administered together is the treatment with the formulation containing the anti-α4β7 antibody. It can be reduced over time during the period. For example, the development of treatment with anti-α4β7 antibody preparations. Treatment with steroids (e.g., prednisone, prednisolone) at the start or before Patients who had been receiving treatment with anti-α4β7 antibody preparations may start steroids as early as 6 weeks later. You will receive a medication plan to reduce the dosage. The steroid dosage will be 4% of the initial dose. Approximately 25% within 8 weeks, and 5% during the gradual dose reduction period between 8-12 weeks of treatment with anti-α4β7 antibody preparations. It will be reduced by 0% and then by 75% in about 12-16 weeks. In one embodiment, anti-α4β7 antibody Steroid administration can be eliminated by approximately 16-24 weeks of treatment with the formulation. Another example So, when starting treatment with anti-α4β7 antibody preparations, or before that, 6-mercaptopurine Patients who were being treated with anti-inflammatory compounds, as mentioned above, were given steroids. Receiving a medication plan that involves reducing the dose of anti-inflammatory compounds, similar to a gradual dose reduction plan. It will become.

[0181] In one embodiment, the method includes administering an effective amount of the formulation of the present invention to a patient. If the body, for example, is in a dry state, the administration process includes the step of converting the formulation into a liquid state. It can be used by injection, for example, intravenous, intramuscular or subcutaneous injection. For use, for example, the dry formulation can be reconstituted with the liquids described above. In another embodiment, the solid or dried formulation may be used, for example, as a patch, cream, or aerosol. Alternatively, it can be administered locally via suppositories.

[0182] This invention relates to the whitening of tissues expressing the molecule MAdCAM (e.g., MAdCAM-1). This also concerns methods for treating diseases related to blood cell infiltration. The methods are available to patients who need them. The present invention comprises administering an effective dose of the anti-α4β7 antibody preparation of the present invention. In the embodiment, the disease is graft It is a host-versus-host disease. In some embodiments, the disease is caused by the molecule MAdCAM (e.g., MAdC White endothelium expressing α4β7 integrin expressing AM-1) It is a disease associated with leukocyte infiltration of tissues as a result of blood cell binding. In other embodiments, The disease may be gastritis (e.g., neutrophilic gastritis or autoimmune gastritis), pancreatitis, or insulin dependence. It is diabetes mellitus. In yet another embodiment, the disease is cholecystitis, cholangitis, or pericholangitis. ru.

[0183] The present invention also relates to a method for treating inflammatory bowel disease in patients. In one embodiment... The method comprises administering an effective amount of the anti-α4β7 antibody preparation of the present invention to a patient. In one embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In another embodiment, Inflammatory bowel disease includes celiac disease, seronegative arthropathy-associated bowel disease, microscopic or corticosteroids. This could be chlorogenic colitis, gastroenteritis (e.g., neutrophilic gastroenteritis), or pouchitis.

[0184] In some embodiments, treatment with anti-α4β7 antibodies alters the CD4:CD8 lymphocyte ratio. No. The CD4:CD8 ratio is measured in blood, lymph node aspirate, and cerebrospinal fluid (CSF). This is possible. CD4 of CSF in healthy individuals + :CD8 + The ratio of lymphocytes is usually about 1 or greater (Svenningsson et al., J. Neuroimmunol. 1995;63:39-46; Svenningsson) et al., Ann Neurol. 1993; 34:155-161). Immunomodulators reduce the CD4:CD8 ratio to less than 1. It can be changed.

[0185] manufactured goods In another embodiment, the present invention provides a pharmaceutical formulation of the present invention and instructions for its use. It is a manufactured product. The manufactured product includes a container. Suitable containers include, for example, bottles, vials. For example, double-chamber vials, vials for liquid formulations with or without needles, with or (with or without needle-free vial of reconstituted solution, vial of solid formulation), syringe (even Examples include double-chamber syringes, pre-loaded syringes, and test tubes. The container is Containers can be formed from various materials, such as glass, metal, or plastic. The container retains the formulation and any labels on or associated with it, and the container indicates instructions for use. Obtain. In another embodiment, the formulation is prepared for self-administration and / or for self-administration. It may contain instructions. In one embodiment, the container holding the formulation is a single-use container. It may be a vial. In another embodiment, the container holding the formulation may be a reusable vial. This involves repeated administration of the formulation using more than one portion of the reconstituted formulation (for example, 2- Allows for 6 doses. The manufactured product also includes other buffers, diluents, fillers, needles, and silicone. The commercial and It may contain other substances that are desirable from the user's perspective.

[0186] Clinical and qualitative analysis In another aspect, the present invention is a method for determining whether a pharmaceutical formulation meets quality standards for a product. The method involves examining the formulation, evaluating its appearance, determining the reconstitution time, and evaluating the lyophilized formulation. To determine the moisture content, measure aggregates in freeze-dried formulations, and measure fragmentation. To do so, measure oxidation / deamidation, and optionally measure biological activity and efficacy. This includes the evaluation of lyophilized pharmaceutical preparations (e.g., humanized anti-α4β7 antibodies) and a predetermined basis Achieving this standard indicates that the product is suitable for clinical use.

[0187] Acceptable quality levels include ≤5.0% moisture, ≤40 minutes of reconstitution time, and pH 6.3 ± 0.3 reconstituted solution, antibody concentration of 54.0-66.0 mg / ml, CEX ≥ 55.0% Major isoforms by SEC, monomers ≥96.0%, high molecular weight ( ≤2.5%) Aggregates), ≥90% H+L chains by SDS-PAGE, 60-140% reference standard adhesion. These are some examples.

[0188] The present invention will be understood more fully by referring to the following examples. However, However, these should not be interpreted as limiting the scope of the present invention. All quotations by Xu are incorporated herein by reference.

[0189] Development protocol for manufacturing formulations A. Anti-α4β7 antibody solution

[0190] Frozen high-concentration anti-α4β7 antibody preparation (vedolizumab, 50 mM histidine, 12 Place a bottle containing 5 mM arginine, 0.06% polysorbate 80 (pH 6.3) at room temperature. Thaw for 16-24 hours. Pour the molten bottle into a stainless steel mixing container and mix. Combine the prepared mixture. Then dilute the mixture with buffer A (50 mM histidine, 125 mM arginine). , 80 mg / ml vedolizuma (0.06% polysorbate 80, pH 6.3) Dilute with b and mix. Then, dilute buffer B (50 mM histidine) containing sucrose. N, 125 mM arginine, 40% sucrose, 0.06% polysorbate 80, Sucrose is added by diluting the preparation at pH 6.3. This step is an anti-α4 The β7 antibody preparation contains 60 mg / ml vedolizumab, 50 mM histidine, and 125 mM Arginine, 10% sucrose, 0.06% polysorbate 80, pH 6.3 liquid Dilute with the formulation.

[0191] B. Freeze drying 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, A vial containing 10% sucrose and a 60 mg / ml anti-α4β7 antibody preparation at pH 6.3. Fill each 20ml glass vial with 5.52ml of liquid into a stoppered vial and freeze-dry. Place in the designated area. Mount the vials on a shelf set to approximately 20°C in a freeze dryer. After installing and closing the door, lower the shelf temperature to approximately -45°C to freeze the solution. At this temperature, 3 After a certain amount of time, the shelf temperature was raised to -20°C for annealing. Annealing took 4 hours. Next, lower the shelf temperature to approximately -45°C and refreeze the solution. Equilibrium the vials at this temperature. Afterward, the air is discharged from the chamber. When the pressure reaches 150 milliliters, the shelf temperature is set to primary. The drying temperature is gradually increased to approximately -24°C. Primary drying involves sublimating all the crystalline ice from the vial. The process continues until the moisture content of the freeze-dried preparation is reduced to approximately 2.5% for secondary drying. Then raise the shelf temperature to 27°C for 16 hours. Once secondary drying is complete, nitrogen gas is added until atmospheric pressure is reached. Fill the chamber and return it. Seal the vial with a stopper and remove it from the freeze-dryer.

[0192] C. Storage and use of lyophilized anti-α4β7 antibodies Frozen vials of anti-α4β7 antibody are stored at -70°C, -20°C, 2-8°C, or 25°C. Store for the desired time. If usable, equilibrate the vial to room temperature. Then, 21 Using a G needle, reconstitute the contents of the vial with a syringe containing sterile water for injection ("WFI"). The amount of WFI is such that the final volume of the reconstituted antibody solution is the same as the volume of the solution before freeze-drying. It is decided to do so. For the pre-freezing volume of 5.52 ml, add 4.8 ml of WFI. Gently rotate the vial, then hold it for 10-30 minutes to reconstitute the formulation, and then... Then, using a syringe, remove the antibody solution and add it to the IV bag or IV infusion for the patient. . [Examples]

[0193] Example Example 1 Comparative data on altering % sugar and amino acid content in freeze-dried formulations. Design an experimental approach to determine the molar ratio of sugars (sucrose and mannitol) and proteins. The effects of changing the ratio, the molar ratio of arginine to protein, and the molar amount of histidine buffer were investigated. It is known that histidine and arginine do not crystallize during the freeze-drying process. In this respect, these are cryoprotective agents or freeze-protective agents. 1.5 ml of the preparation is used in 5 ml of Fill into vials, freeze-dry, pre-dry at -30°C with 150 mlitol, then at 20°C. Secondary drying was performed with 50 milliliters. After various storage conditions, the freeze-dried solution was reconstituted into 1.5 ml portions. The stability of the formulation is shown in Tables 1-3 (edited results for 60 mg / ml from two experiments). Figure 6 A is stored at 40°C, and the monomer ratio is obtained when the pH and the molar ratio of sugar to arginine are changed. This document presents a predictive model for changes in aggregate ratio and major isoform ratio. The drug's stability was best at a low pH and with a high molar ratio of (sugar + arginine) to protein. The molar amounts of histidine examined did not affect the stability of the formulation. All of them contained 1-2% moisture during storage.

[0194] [Table 1]

[0195] [Table 2]

[0196] [Table 3]

[0197] Figure 6A shows a predictive model based on a statistical analysis of the 40°C data from Tables 1-3. The model for the change in monomer ratio per month at 40°C, based on SEC analysis, is -3.10. +(0.386) * pH +0.000516 * ((moles of sugar + moles of arginine) / tan This is the mole of protein. Regarding the change in aggregate ratio per month at 40°C using SEC analysis... The model is 2.43-(0.263) * pH -0.000787 * ((Moles of sugar + alcohol This is calculated as (moles of ginine) / (moles of protein). The monthly amount of ginine at 40°C is calculated by CEX analysis. The model for the change in the required isoform ratio is -2.54 + (0.109). * pH-0 .00130 * This is ((moles of sugar + moles of arginine) / moles of protein). The line shows the results for the predictive model, and the outer line represents the 95% confidence interval for the predictive model. show.

[0198] Figure 6B shows the input factors pH, sugar:protein molar ratio and arginine:protein molar ratio. Tables 1-3 show alternative models based on statistical analysis of data at 40°C, in the case of a ratio. The model for the change in monomer ratio per month at 40°C using SEC analysis is -3.0 2 + (0.370) * pH +0.000482 * ((moles of sugar) / (moles of protein)) )+0.000657 *This is (moles of arginine / moles of protein). SEC analysis The model for the change in the monthly aggregate ratio at 40°C is 2.35-(0.244 ) * pH -0.000727 * ((moles of sugar) / (moles of protein))-0.0010 2 * (Moles of arginine / Moles of protein). Monthly amount at 40°C according to CEX analysis. The model for the change in the ratio of major isoforms is -2.92 + (0.210). * pH +0.00164 * ((moles of sugar) / (moles of protein))-0.000220 * This is (moles of arginine / moles of protein). The center line represents the results for the predictive model. The outer line indicates the 95% confidence limit for the predictive model.

[0199] Example 2 Stability data Stability after storage under the instructed storage conditions (5°C and 25°C / 60%RH for up to 24 months) The stability of the formulation was examined using three primary stability batches (Batches A, B, and C). All are frozen, identical liquid formulations: 60 mg / ml anti-α4β7 antibody, 50 mM Histidine, 125 mM arginine, 10% sucrose, 0.06% polysorbate It contains 80% pH and 6.3. For batch A, 3.5 ml of the solution is mixed with 20 ml of the solution. Fill the bottles, freeze-dry, and for batches B and C, add 20 ml of 5.52 ml of solution. The mixture was filled into vials and freeze-dried.

[0200] In another study, 60 mg / ml anti-α4β7 antibody, 50 mM histidine, 125 mM Arginine, 10% sucrose, 0.06% polysorbate 80, pH 6.3 Each drug formulation was freeze-dried in two volumes, 3.5 ml and 9.5 ml, to obtain a stability sample. Batch R and S values ​​were obtained and analyzed over 38 months. The blank was NT( (Not tested)

[0201] The data (Tables 4-19) is for up to 38 months at 5°C and up to 30 months at 25°C / 60%RH. The study demonstrated that the antibody preparations remained stable when stored. All products were tested at 38 months. This is likely because it remained within the specifications for a considerable period.

[0202] [Table 4]

[0203] [Table 5]

[0204] [Table 6]

[0205] [Table 7]

[0206] [Table 8]

[0207] [Table 9]

[0208] [Table 10]

[0209] [Table 11]

[0210] [Table 12]

[0211] [Table 13]

[0212] [Table 14]

[0213] [Table 15]

[0214] [Table 16]

[0215] [Table 17]

[0216] [Table 18]

[0217] [Table 19]

[0218] Cation exchange chromatography (CEX) High-performance liquid chromatography (HQLA) with a weak cation exchange column for phosphate / sodium chloride The charge species in anti-α4β7 antibody preparations are separated using the gradient, and the charge composition of the antibody species is measured. The acidic isoform elutes before the main isoform, and then the main isoform elutes. Basic isoforms are eluted.

[0219] Stability data for all batches of vedolizumab generated using the CEX assay. The results are shown in Tables 3, 6-8 and 14-16. The table shows that under these storage conditions, the percentage is below 55%. This indicates that there was no tendency to decrease the proportion of the main isoforms.

[0220] Size exclusion chromatography (SEC) Analytical SEC columns (Tosoh Bios, King of Prasha, Pennsylvania) SEC is performed using cience, LLC. The mobile phase is phosphate-buffered saline solution. Yes, and absorption is monitored at 280nm.

[0221] Stability data generated using the SEC assay are presented in Tables 1, 2, 4, 5, 12, and 13. The table shows that all of the listed storage conditions resulted in a decrease in monomer ratio below 96.0%. This indicates that it was not present in all batches under all of the listed storage conditions. The aggregate ratio remained at ≤2.5%.

[0222] SDS-PAGE assay Under reducing conditions, the concentration is 4-20%, and under non-reducing conditions, it is 4-12%. SDS-PAGE was performed using Tris / glycine gel in Carlsbad, California. The reconstituted antibody preparation is diluted with liquid formulation buffer, and then 10% 2-mercaptophosphate is added. With ethanol (reducing sample buffer) or without 2-mercaptoethanol (non-reducing) Sample buffer) Tris / glycine SDS sample buffer (2X, Invitrogen) 1: Dilute to 2. Briefly heat the sample and compare it with the molecular weight marker (Invitrogen). Apply the colloidal Coomassie Blue (Invitro) according to the manufacturer's instructions. The gel is stained with (gen). Protein bands are analyzed by concentration measurement and reduction. In gels, the percentages of heavy and light chains are identified, and in non-reducing gels, the percentage of IgG is identified.

[0223] Stability data generated using the reduced SDS-PAGE assay are shown in Tables 9 and 17. The ratio of heavy chain + light chain (H + L) is as follows under all the storage conditions listed for the stable lot. No significant changes were observed. The band pattern was similar to the reference standard, and %(H+L) was It remained at the ≥90% level.

[0224] Binding effectiveness HuT78 cells (human) suspended in PBS, 0.01% sodium azide, and 1% BSA. T-cell lymphoma cells, American Type Culture Collection, Manassas, Virginia Contact the primary test antibody (which has been serially diluted) with the (amount) sample. After incubation on ice, the cells are then... Wash and treat with a fluorescently labeled secondary antibody. After further washing, fix the cells and use a fluoroscopy solution. Itometry (Becton Dicki, Franklin Lakes, New Jersey) It is suspended in FACS reagent for analysis by nson. Also, U.S. 7,147 See also issue 851.

[0225] The binding efficacy of vedolizumab was measured compared to a reference standard, and the % reference standard and EC50 were expressed as follows: Reported. Stability data is presented in Tables 10 and 18. % Data regarding reference standards may vary. Although it showed some activity, it remained within the specified limits under all storage conditions. The evaluation of vedolizumab... The selected lots did not show a tendency for reduced binding efficacy under the listed storage conditions.

[0226] According to Karl Fischer, moisture For the Karl Fischer method of determining moisture content, the formulation is titrated with methanol. The data is presented in Tables 11 and 19. Vedolizumab was evaluated under all of the listed storage conditions. All lots had a moisture content of less than 5%.

[0227] Capillary isoelectric focusing (cIEF) iCE280 Full Column Detection cIEF System (Conver, Toronto, Ontario) cIEF will be performed using gent Biosciences. Amphoteric electrolytes will be provided by the manufacturer. Therefore, it can be selected as recommended, and commercially available amphoteric electrolyte combinations are possible. Useful combinations include 3-10 and 5-8 PHARMALYTE(trademark) (New Zealand This is GE Healthcare in Piscataway, Jersey.

[0228] Example 3: Modeling the scaling up of the freeze-drying process Quality build-up techniques were used while manipulating the load and solid content of the formulation in the freeze-dryer. The values ​​changed by 33-100%. The load levels were 0.5×, 1.0×, and 1.5× for the target formulation. The inclusion of the formulation altered the solid content of the formulation by 9-27%. These formulations were similar to T g It had a higher solid content ratio, and the primary drying time increased. In addition, Even larger R in solid content p The product temperature rose due to the load. The load also affected both stages of drying. This has an effect (Figure 8).

[0229] Example 4: Non-clinical safety test Natalizumab and vedolizumab for immune surveillance mechanisms in the CNS in rhesus monkey EAEs The trial was designed to compare the effects of the two drugs. Eight animals received a placebo control once a week. Seven animals were administered 30 mg / kg of natalizumab once a week. Zumab was administered once a week to 7 animals. Clinical symptoms of EAE were observed: flow cytometry - Measuring the frequency and ratio of leukocyte subsets in CSF; using MRI to examine the brain The total lesion volume in T2 was measured; and the lesion volume and demyelination of the brain were determined using histopathological methods.

[0230] Vedolizumab did not delay the onset of clinical symptoms of EAE compared to placebo control. Natalizumab did not suppress the onset of EAEs, nor did it reduce the magnitude of clinical scores. Compared to placebo control, it significantly delayed the onset of clinical symptoms of EAE (p<0.05). This reduced the incidence of EAE and the magnitude of clinical scores (Figure 9).

[0231] Vedolizumab is used to treat white blood cells and T lymphocytes (helper T lymphocytes, cytotoxic T lymphocytes). It did not suppress CSF infiltration by B lymphocytes, natural killer cells, or monocytes. In contrast, natalizumab inhibited CSF infiltration.

[0232] Vedolizumab is detected by increased T2 and decreased MTR via MRI. It did not inhibit the accumulation of brain lesions. Natalizumab did not inhibit disease deformation in all but one animal. It hindered development. Significant (p<0.05) inhibition of brain infiltration and demyelination was measured by histology. It was decided.

[0233] Vedolizumab administered in vivo and anti-α4β7 monoclonal for analysis added in vitro. As demonstrated by competitive binding assays between Nal antibodies, α4β7 integrands are under investigation. Phosphorus was saturated with vedolizumab. Anti-α4β7mAb for analysis was vedolizumab. In administered animals, it does not bind to memory helper T cells. Therefore, in the CNS, The lack of effect of mab is due to the biological properties of α4β7 integrin's gastrointestinal targeting.

[0234] In summary, vedolizumab (an α4β7 antagonist) does not inhibit EAE. In contrast, Natalie Zumab (an antagonist of α4β1 and α4β7) inhibits EAE. α4β1 integrin inhibits E It is involved in CNS infiltration in AEs. Therefore, vedolizumab is involved in α4β1 integrin It does not antagonize and does not damage the immune surveillance mechanism of the CNS in the EAE of rhesus monkeys, therefore The risk of patients developing PML may be lower than with lizumab.

[0235] Example 5: Phase I clinical trial with vedolizumab 49 healthy subjects were randomized and given a single dose of the study drug: 39 subjects were affected by Vedoliz Mab (5 mg / ml antibody, 20 mM citrate / citric acid, 125 mM sodium chloride) Um, 0.05% polysorbate 80, pH 6.0 (-70°C long-term, -20°C 3 months) Ten subjects received a placebo (which was stored for a month). Of the 39 people who received the drug, 8 received doses of 0.2, 2.0, 6.0, and 10.0 mg, respectively. Seven people received vedolizumab at a dose of 0.5 mg / kg. All 49 people completed the trial. Done.

[0236] Significant differences in demographic or baseline characteristics were observed in the vedolizumab cohort. There was none. The average age ranged from 35.4 to 51.0 years; individual subjects ranged from 21 to 63 years old. It was successful.

[0237] Penalty shootout results Vedolizumab was administered as an intravenous infusion of 0.2–10.0 mg / kg over 30 minutes. The values ​​of Cmax and the area under the serum drug concentration-time curve (AUC) increased with increasing dose. The fact that the dose-adjusted Cmax is nearly identical across the cohort means that this parameter The dose-proportional relationship of the drug was demonstrated. The dose-normalized area under serum drug concentration values ​​from zero to infinity ( AUC 0-∞ The fact that it increased as the dose increased up to 2.0 mg / kg means In this study, the AUC increased with increasing doses across a low range of doses administered. 0-∞ in This indicates a non-linear increase. Subsequently, AUC 0-∞ It increases in proportion to the dose. This means that the AUC over the dose range of 2.0 to 10.0 mg / kg 0-∞ Linear It indicates sex. AUC 0-∞ The increase in was 10.0 compared to the 0.2 mg / kg dose. The levels were approximately 2 to 4 times higher than expected for the mg / kg dose.

[0238] Similarly, the estimated clearance, volume of distribution, and final half-life are 0.2–2.0 mg / kg It was dose-dependent across the dose range. As the dose increased, clearance decreased. The volume of distribution increased, and as a result, the final half-life was extended. However, from 2 to 10.0 Up to mg / kg, there was no clear change in these parameters, but this is because at low concentrations... This suggests saturation of the rapid excretion process of vedolizumab. Furthermore, a slower linear excretion process is... Furthermore, this is likely to be the main factor in the large clearance fraction of vedolizumab at higher doses. stomach.

[0239] In some subjects who developed HAHA in response to vedolizumab, H occurred within the range of each dose level. Vedolizumab showed faster clearance compared to AHA-negative subjects.

[0240] [Table 20-1] [Table 20-2]

[0241] C max After reaching this point, the serum concentration of vedolizumab reaches approximately 1-10 mg / l. The concentration generally decreased exponentially until a certain point. After that, it was thought that the concentration would decrease non-linearly. .

[0242] C max The AUC values ​​increased with increasing dose. For available data, see below. , dose-corrected C max The fact that this was nearly identical across cohorts means that this paragraph This shows the dose-proportionality of the meter. Dose-normalized area (AUC) 0-∞ up to 2.0 mg / kg The fact that it increased with increasing dose suggests that in the lower range of doses administered in this study... AUC with increasing dose 0-∞ This indicates that there was a nonlinear increase in [location]. Subsequently, AUC 0-∞ The fact that it increased in proportion to the dose means that 2.0-10.0 mg AUC over the dose range of / kg 0-∞This demonstrates linearity. AUC 0-∞ in The increase was greater than what would be expected at the 10.0 mg / kg dose compared to the 0.2 mg / kg dose. It was about 2 to 4 times more expensive.

[0243] PD results In a cohort, vedolizumab was administered via intravenous infusion over 30 minutes at doses of 0.2–10.0 mg / kg. The following table shows the PD parameters for vedolizumab for Act-1 and MAdCAM, respectively. This is summarized in Section 21 and Table 22.

[0244] [Table 21]

[0245] [Table 22]

[0246] Vedolizumab is used to measure PD parameters at all time points when vedolizumab can be measured in serum. Vedolizumab concentration inhibited Act-1 and MAdCAM-1-Fc to the near maximum extent. Once the levels fall below the detection limit of the assay, Act-1 and MAdCAM-1 -Fc inhibition recovered to almost baseline levels.

[0247] In some subjects who developed HAHA to vedolizumab, HAHA-negative results were observed at each dose level. A rapid loss of α4β7 receptor saturation was observed compared to sexual partners.

[0248] Safety results Vedolizumab is generally safe and well-received with a single IV dose of 10.0 mg / kg. During the trial, no deaths, serious adverse events (SAEs), or AEs that would lead to the discontinuation of the trial occurred. Ta.

[0249] Immunogenicity / Formation of human anti-human antibodies (HAHA) One person (10%) in the placebo group and 21 people (54%) in the combination vedolizumab group Some of the samples at certain points in the study were positive for HAHA. Positive HAHA samples were found in all of the administered cohorts. However, HAHA titers >125 were observed only in the two lowest dose vedolizumab groups. Dose-dependent suppression of HAHA formation had previously been observed with vedolizumab. Of the 22 patients treated with vedolizumab, 19 currently have neutralized HAHA.

[0250] [Table 23]

[0251] One patient in the placebo group and eleven patients in the vedolizumab group remained HAHA-positive.

[0252] [Table 24]

[0253] conclusion This Phase I trial evaluates the PK / PD and initial safety of vedolizumab derived from CHO cells. Sexual characteristics were characterized. The results of this study were used in an important Phase 3 trial for inflammatory bowel disease. This assisted in selecting the appropriate dosage.

[0254] Vedolizumab showed dose-proportionality across the dose range investigated for the Cmax parameter. As shown, the dose-dependent changes in AUC0-inf, CL, Vz, and t1 / 2 were 0.2 The fact that this was observed at ~2.0 mg / kg suggests a nonlinear pharmacokinetic behavior of vedolizumab. At dose levels greater than 2.0 mg / kg, these parameters are further No changes were observed, suggesting saturation of the rapid excretion process of vedolizumab at low concentrations. Furthermore, the slower linear excretion process is necessary for the clearance of vedolizumab at even higher doses. This is likely the main factor in large fractions.

[0255] Vedolizumab was at its highest level at all time points when vedolizumab could be measured in serum. Alternatively, inhibit PD parameters, Act-1, and MAdCAM-1-Fc at near-maximum levels. When vedolizumab once falls below the detection limit of the assay, Act-1 and The inhibition of MAdCAM-1-Fc was restored to almost baseline levels.

[0256] In some subjects who developed HAHA to vedolizumab, H was observed within the range of each dose level. Vedolizumab showed faster clearance and faster α4β7 receptor saturation compared to AHA-negative subjects. Loss was recognized.

[0257] Vedolizumab was well-received. During the study, there were no deaths, SAEs, or discontinuation of the study drug. No adverse events occurred, and no dose-to-toxicity relationship was observed. Systemic opportunistic infections ( No malignancies (including PML) or other malignant tumors were reported.

[0258] Unlike nonspecific α4 antagonists, vedolizumab causes lymphocytosis or circulating eosinusitis. There was no correlation with an average increase in lymphocytes, basophils, or monocytes, nor was there any evidence of lymphocyte depletion. Ta.

[0259] Vedolizumab induced HAHA formation, but the highest titer (>125) was the lowest of two types. This was observed only in the dose group, which supports previous findings of dose-dependent reduction in immunogenicity. These findings indicate that high doses of vedolizumab are clinically significant in HAHA. This indicates that the formation of [the substance] should be suppressed as much as possible.

[0260] In conclusion, vedolizumab is generally safe and can be administered to healthy subjects at a dose of 0.2–10.0 mg / kJ. When administered as a single dose of g, it was well tolerated.

[0261] Example 6: Determination of the effect of vedolizumab on the CD4:CD8 ratio Reconstituted from a lyophilized 10% sucrose preparation, diluted in a 0.9% saline infusion system. Healthy subjects aged 18-45 years were treated with a single 450 mg dose of vedolizumab. Lumbar puncture was performed before (baseline) and 5 weeks after a single 450 mg dose of dolizumab to examine the brain. Cerebrospinal fluid was collected. Each subject served as a control group.

[0262] MS patients treated with natalizumab showed improved CSF CD4+:CD after just one dose. Previous studies demonstrated an effect on the ratio of 8+ lymphocytes and a reduction in the number of brain lesions. (Stuve et al. Arch Neurol. 2006; 63: 1383-1387; Stuve et al. Ann Neurol. 2006;5 9:743-747. Based on Miller et al. N Engl J Med. 2003;348(1):15-23), and also Bedli A 450 mg dose of zumab at 5 weeks was sufficient to saturate the target, and 300 mg every 4 weeks was sufficient. Providing serum concentrations exceeding the estimated steady-state trough level related to the Phase 3 dosing plan for mg Since I was providing the sample, I chose the 5-week mark.

[0263] Approximately 15 ml of CSF was obtained from each subject for immunological phenotyping. The following criteria were used: RBCs / μl ≤10 per sample (to minimize peripheral blood contamination); negative C SF culture results; appropriate T lymphocyte count in each flow cytometry sample; and Bedliz CSF samples were included in the analysis if they met the condition that no serum antibodies against mab were detected.

[0264] Median serum vedolizumab concentration at week 5 (34.80 μg / ml) and individual subject serum vedolizumab concentration (2 The range of 4.9 to 47.9 μg / ml is the estimated baseline for the Phase 3 dosing regimen. The concentration was higher than the normal trough concentration (approximately 24 μg / ml). This indicates a high level (>90%) of α4. β7 receptor saturation was observed at week 5, as measured by MAdCAM-1-Fc. This indicates that the target vedolizumab was saturated at the time of the endpoint evaluation.

[0265] Vedolizumab was not detected in any CSF samples. Output limit=0.125μg / m l)

[0266] Effects of CD4+ and CD8+ T lymphocyte count and ratio Vedolizumab did not significantly reduce the CD4+ to CD8+ ratio (Table 25). No subjects had a CD4+ to CD8+ ratio of <1 (p<0.0001, one-sided). (t-test). Vedolizumab significantly reduced the number of CD4+ and CD8+ T lymphocytes in CSF. In addition, there were no significant changes in %CD4+ and %CD8+ T lymphocytes in the CSF. There were none (Table 26). Also, peripheral blood WBC, CD4+ and CD8+ memory T lymphocytes No significant changes were observed (Table 27).

[0267] [Table 25]

[0268] [Table 26]

[0269] [Table 27]

[0270] summary Vedolizumab, after a single dose of 450 mg, improved CSF CD4+ and in healthy volunteers. CD8+ cell count or CD4 + and CD8 + It did not affect the ratio. After administration, CS No subjects in F showed a decrease in the CD4+:CD8+ ratio to less than 1. Vedolizumab was used in CS. It was not detected in F. In addition, CD4 of total WBCs or memory T lymphocytes in peripheral blood. No changes were observed in the + and CD8+ subsets. Target (α4β7) in blood. Saturation of ) occurred in all subjects at the time of the endpoint evaluation. Phosphorus of CD4+ and CD8+ in CSF The levels and ratios of pacyles were similar to those previously reported in the literature.

[0271] These results suggest a dual approach to the physiological immune surveillance mechanisms of the CNS and pathological inflammation of the CNS in monkeys. This is consistent with the lack of effect of vedolizumab (see Example 4).

[0272] Example 7: Long-term clinical experience with vedolizumab for the treatment of IBD Phase 2 open-label safety extension study completed to evaluate the long-term pharmacokinetics (PK) of vedolizumab. The pharmacodynamics (PD), safety, and efficacy were evaluated. The patients ranged in age from 18 to 75 years and had ulcerative colitis. Have you previously participated in a previous PK / PD safety trial in patients with colitis, or are you 36? Within a month of screening, by endoscopy and / or histopathology and / or radiology The patient had been experiencing symptoms of IBD for at least two months.

[0273] All patients received vedolizumab at 2 mg / kg or 6 mg / kg (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 storage, and at -20°C for up to 3 months) 1, 15 And on day 43, and thereafter every 8 weeks until a total of 78 weeks. The patient received treatment. Patients with ulcerative colitis or Crohn's disease without symptoms, or those who have previously participated in clinical trials He was a patient with ulcerative colitis.

[0274] Effectiveness / Quality of Life (QOL), Partial Mayo Score (PMS), Crohn's Disease Activity Index The results of the study were evaluated using the CDAI and the Inflammatory Bowel Disease Questionnaire (IBDQ). .

[0275] Penalty shootout results The average pre-infusion vedolizumab concentration was dose-proportional and remained fixed throughout the study. It was detectable through this method.

[0276] PD results Throughout the entire study, receptor (%ACT-1+[CD4+CD45R) was observed at all dose levels. O-high and %MADCAM+[CD4+CD45RO high] were almost completely inhibited.

[0277] Partial Mayo score The mean baseline PMS was better treated than in patients with recurrent ulcerative colitis (2.3). Patients with ulcerative colitis who had not experienced this condition had a higher (5.4) score. By day 43, PMS was recurring. A significant reduction was observed in both patients with relapsed ulcerative colitis and patients with ulcerative colitis who had never received treatment. 15 By day 5, the mean scores of the two groups were similar. The mean PMS decreased until day 267. It continued, and then it became flat.

[0278] Crohn's disease activity indicators CDAI in CD patients decreased from 294.6 at baseline to 237.7 at day 43. The level continued to decline until day 155 (156.1).

[0279] IBDQ Patients with recurrent ulcerative colitis had the highest mean IBDQ score at baseline. By day 43, the score had increased in all three disease groups. The mean IBDQ score was the same in all three disease groups. The number of cases continues to increase over time, and in Crohn's disease patients, on day 155, the number of cases of ulcerative colitis that had not been treated was higher. In patients with colitis and those with recurrent ulcerative colitis, the peak was reached on day 491.

[0280] C-reactive protein Patients with recurrent ulcerative colitis and Crohn's disease show a decrease in mean C levels up to day 155. The RP level was observed and then flattened out. Untreated ulcerative colitis patients, ulcers Patients with recurrent colitis had lower mean CRP levels at baseline (2.28 (vs. 7.09). The mean CRP levels of patients with ulcerative colitis who had never received treatment were assessed. It remained relatively constant at all points in time.

[0281] Other safety results During the trial, no systemic opportunistic infections (including PML) were reported. One patient had a single infection. At one point in time, the patient tested positive for JC viremia, but at all other points in time, they tested negative for JC viremia. (72 people) Three of the patients (4%) tested positive for HAHA (two of these were temporarily positive). The study investigated liver toxicity, lymphocytosis, lymphopenia, or other drug-related adverse reactions. They failed to provide evidence of changes in clinical test values.

[0282] conclusion Vedolizumab administered once every 8 weeks at a dose of 2.0 or 6.0 mg / kg up to 78 weeks , achieving saturation of target receptors, resulting in a long-lasting average reduction in disease activity and improved IBD It was accompanied by Q and generally safe, well-received, and exhibited tolerable immunogenicity.

[0283] Example 8: Induction of response and remission in patients with moderate to severe active Crohn's disease. A randomized, double-blind, placebo-controlled, multicenter trial was completed, with two measurements taken at week 6 (weeks 0 and 2). In patients who failed with TNFα antagonists (after administration) and at 10 weeks (after 3 doses), 30 0 mg dose (freeze-dried 50 mM histidine, 125 mM arginine, 0.06%) 60 mg / ml antibody in polysorbate 80, 10% sucrose, pH 6.3 The inductive effect of vedolizumab (reconstituted from the formulation) was evaluated. The trial involved 416 patients. Of these, 75% were patients who had failed with TNFα antagonists, and 25% were patients who had failed with TNFα. There was no prior drug experience. Demographics and medication for associated IBD were balanced across the entire treatment group. We took the data. The baseline disease characteristics, excluding baseline disease activity, were also analyzed across the entire treatment group. A balance was maintained across the board.

[0284] The primary endpoint specified in the trial was remission (%) at 6 weeks in the group that failed with TNFα antagonists. The key secondary endpoint to be evaluated (in the sequential trial procedure) was remission at 6 weeks for the entire population. (%), remission at 10 weeks in the group that failed with TNFα antagonists and in the overall group (%) (Hoc (Using the Hberg method) 6 weeks and 10 weeks in the group that failed with TNFα antagonists and in the entire group. Sustained remission (%) (using the Hochberg method), and failure with TNFα antagonists The improved response (%) was observed in the group at week 6.

[0285] [Table 28]

[0286] [Table 29]

[0287] [Table 30]

[0288] [Table 31]

[0289] The trial showed that patients who failed with TNFα antagonists required three doses to induce remission. The study showed that the remission rate in patients who failed with TNFα antagonists increased between weeks 6 and 10. However, this was only the case for the vedolizumab group (not placebo). Administration of TNFα antagonists. For patients with no prior experience, the remission rate did not substantially increase between weeks 6 and 10. Among patients with severe disease who failed with TNFα antagonists, 43% were TNFα antagonists They never responded to the drug, and 45% lost their response.

[0290] Example 9: Induction and maintenance of response and remission in moderate to severely active Crohn's disease To evaluate the induction and maintenance of response and remission in patients with moderate to severe active ulcerative colitis. To achieve this, we designed single-stage trials, including two randomized, double-blind, multicenter trials. Demographic and Baseline disease characteristics were similar across all treatment groups.

[0291] Induction studies using intravenous administration were performed with 50 mM histidine, 125 mM arginine, and 0 60 mg / ml at 0.06% polysorbate 80, 10% sucrose, pH 6.3 A 300 mg dose reconstituted from the lyophilized antibody was administered 6 times after two doses of vedolizumab. At the weekly endpoint, vedolizumab was compared to placebo.

[0292] Maintenance studies using the same formulation and route as the induction studies were compared to placebo against vedolizumab. However, vedolizumab was administered every 4 weeks, while placebo was administered every 8 weeks. The drug was administered. Each patient was between 18 and 80 years old and had moderate to severe active ulcerative colon. Diagnosed and having received minimal conventional treatment (e.g., corticosteroids) over the past 5 years Although they showed an inappropriate response, loss of response, or nonacceptance to at least one of the conditions for IBD, Patients may be receiving conventional treatment. The endpoint of this trial is 52 weeks, and the guided response The group of participants was analyzed. Both phases of the experiment were analyzed at their primary endpoint, i.e., the clinical endpoint in the induction. The patient achieved both a palliative response and clinical remission during maintenance.

[0293] Blood samples were collected and the concentration of vedolizumab during the test was measured. The mean serum concentration of lizumab was 20–30 μg / mL. This was observed during the 30-minute period following a 300 mg dose. The mean trough serum concentration of vedolizumab at steady state after IV infusion was determined in the q8wks dosing plan. The dosage is between 9 and 13 μg / mL, and in the q4wks medication plan, it is between 35 and 40 μg / mL. Yes, it was found. The median plasma concentration of vedolizumab at the end of the infusion was in the q8wks (8-week) medication plan. The values ​​were between 98 and 101 μg / mL, and in the q4wks (4-week) medication plan, the values ​​were 129 and 13. The level was around 7 μg / mL.

[0294] Tables 32-35 provide summaries of the response to induction and maintenance studies. A significantly larger group of patients showed clinical response, remission, and mucosal healing at 6 weeks compared to placebo. This was achieved (Table 32). 39% of the inclusive analysis population in the induction phase had failed previous anti-TNF-α therapy. The clinical response and remission rates were high in patients with a history of failure with anti-TNF-α therapy. Vedolizumab was more effective than placebo in both patients who were not exposed to anti-TNF-α and those who were not. Preliminary analysis over 6 weeks showed adverse events (AEs), serious AEs, and the study's The rate of adverse events leading to discontinuation was higher in the placebo group than in the vedolizumab group. The vedolizumab patient group also showed significantly higher rates of clinical remission, mucosal healing, and coagulation at 52 weeks. Luticosteroid-free remission and long-lasting response and remission were achieved (Table 33). Maintenance study 32% of this group had a history of failure with previous anti-TNF-α therapy. Clinical remission and long-lasting responses The response rate was higher than placebo in both patients who had failed TNF and those with no prior TNF experience. The risk was higher for dolizumab. In the safety population (N=895) at 0-52 weeks, adverse events were observed. (AEs), severe AEs and severe infections were similar between the vedolizumab group and the placebo group. No increase in the rate of opportunistic infections or enteric infections was observed in the vedolizumab group.

[0295] [Table 32]

[0296] [Table 33]

[0297] [Table 34]

[0298] [Table 35]

[0299] Example 10: Induction and maintenance of response and remission in patients with moderate to severely active Crohn's disease. This study evaluates the induction and maintenance of response and remission in patients with moderate to severely active Crohn's disease. Therefore, we designed single trials, including two randomized, double-blind, multicenter trials. Demographic and veterinary considerations were taken into account. The disease characteristics in the line of treatment were similar across all treatment groups.

[0300] Induction studies using intravenous administration were performed with 50 mM histidine, 125 mM arginine, and 0 60 mg / ml at 0.06% polysorbate 80, 10% sucrose, pH 6.3 A 300 mg dose reconstituted from the lyophilized antibody was administered 6 times after two doses of vedolizumab. At the weekly endpoint, vedolizumab was compared to placebo.

[0301] Maintenance studies using the same formulation and route as the induction studies were compared to placebo against vedolizumab. However, vedolizumab was administered every 4 weeks, while placebo was administered every 8 weeks. The drug was administered. The endpoint of this trial was 52 weeks, and the population of induced responders was analyzed.

[0302] Surprisingly, this trial yielded very similar results in the groups at weeks Q4 and Q8. This was shown. Tables 36-39 provide summaries of the responses to the induction and maintenance tests. Compared to placebo. A significantly larger group of patients treated with vedolizumab achieved clinical remission and a high response rate. (Table 36). The ratio of clinical remission and high response was compared to patients who had previously failed with TNF and In both groups with no prior TNF exposure, the levels were higher in vedolizumab patients than in placebo patients. The rates of adverse events (AEs), serious AEs, and serious infections were different in the vedolizumab group and the placebo group. Similarities were observed in both groups. An increased rate of opportunistic infections or enteric infections was observed in the vedolizumab group. I couldn't do it.

[0303] [Table 36]

[0304] [Table 37]

[0305] [Table 38]

[0306] [Table 39]

[0307] [Table 40]

[0308] The present invention has been particularly shown and described with reference to its preferred embodiments, but appended Without departing from the scope of the present invention as encompassed by this invention, various modifications in form and detail may be made. Those skilled in the art will understand what can be accomplished within that context.

[0309] The following are examples of aspects of the present invention. [1] A stable formulation comprising a non-reducing sugar, an anti-α4β7 antibody, and at least one free amino acid The formulation contains a mixture of acids, and the formulation is in solid form, with a molar ratio of non-reducing sugar to anti-α4β7 antibody ( A stable formulation with a mol:L ratio exceeding 600:1. [2] The formulation according to [1], further comprising a buffering agent. [3] The non-reducing sugars include mannitol, sorbitol, sucrose, trehalose and so A formulation selected from the group consisting of these combinations [1]. [4] The free amino acids are histidine, alanine, arginine, glycine, glutamic acid A preparation described in [1], selected from the group consisting of nic acid and combinations thereof. [5] The formulation according to [1], further comprising a surfactant. [6] The surfactant is polysorbate 20, polysorbate 80, poloxamer and A formulation selected from the group consisting of combinations thereof [5]. [7] The formulation is freeze-dried, and before freeze-drying, at least about 5% to about 10% of anti-α4β7 A preparation containing an antibody as described in [1]. [8] The formulation contains at least about 6% anti-α4β7 antibody before lyophilization [1] A formulation of [the substance]. [9] The formulation contains free amino acids between approximately 100 mM and approximately 175 mM [1]. The listed preparation.

[10] The formulation according to [1], wherein the formulation is a liquid formulation.

[11] The formulation according to [1], wherein the formulation is a dried formulation.

[12] The formulation described in [1] is stable for at least 3 months at 40°C and 75%RH. formulation.

[13] A stable liquid formulation comprising a non-reducing sugar and an anti-α4β7 antibody in aqueous solution and at least It contains one free amino acid, and the molar ratio (moles:moles) of non-reducing sugar to anti-α4β7 antibody is 60. A stable liquid formulation with a ratio exceeding 0:1.

[14] The liquid formulation according to

[13] , wherein the formulation further comprises a buffering agent.

[15] The non-reducing sugars include mannitol, sorbitol, sucrose, trehalose and A liquid formulation as described in

[13] , selected from the group consisting of combinations thereof.

[16] The free amino acids include histidine, alanine, arginine, glycine, gluta A liquid formulation as described in

[13] , selected from the group consisting of mic acid and combinations thereof.

[17] The liquid formulation according to

[13] , wherein the formulation further comprises a surfactant.

[18] The surfactant is polysorbate 20, polysorbate 80, poloxamer and A liquid formulation as described in

[17] , selected from the group consisting of combinations thereof.

[19] The liquid formulation according to

[13] , wherein the formulation has a pH between approximately 5.5 and approximately 7.5.

[20] The liquid formulation according to

[13] , wherein the formulation has a pH between approximately 6.0 and approximately 6.5.

[21] The preparation contains at least about 60 mg / ml to about 80 mg / ml of anti-α4β7 antibody A liquid formulation as described in

[13] , including the following:

[22] The preparation contains at least about 60 mg / ml of anti-α4β7 antibody

[13] A liquid formulation.

[23] A stable formulation containing at least approximately 60 mg / ml to approximately 80 mg / ml of anti-α4 The formulation contains a β7 antibody, a buffering agent, and at least about 10% (w / w) of sugar, and the formulation is a liquid formulation. A stable formulation.

[24] The formulation according to

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

[25] The formulation according to

[23] , wherein the sugar is sucrose.

[26] A stable formulation containing at least approximately 60 mg / ml of anti-α4β7 antibody and at least The formulation also contains approximately 10% (w / w) of non-reducing sugars, and is a stable formulation that can be freeze-dried.

[27] The formulation according to

[26] , wherein the formulation further comprises polysorbate 80.

[28] The formulation according to

[26] , wherein the non-reducing sugar is sucrose.

[29] A stable formulation comprising a non-reducing sugar, an anti-α4β7 antibody, histidine, arginine, and The formulation contains a mixture of resorbate 80, and the formulation is in solid form, and contains non-reducing sugars and anti-α4β7 anti- A stable formulation with a molar ratio (moles:moles) exceeding 600:1.

[30] The formulation further comprises a metal chelating agent as described in any of

[13] to

[25] . formulation.

[31] The preparation according to any one of

[13] to

[25] , wherein the preparation further comprises an antioxidant.

[32] A formulation according to any one of [1] to

[31] , wherein the antibody is vedolizumab. A method for producing a preparation described in any one of items

[33] [1] to

[32] , the said method A method that includes maintaining a product temperature below the decay temperature during primary drying.

[34] The method of

[33] further comprising an annealing step.

[35] A method for determining the quality of a freeze-dried preparation, wherein the preparation is examined by appearance To investigate, to determine the reconstruction time, to determine the moisture content, and to determine the presence of condensation. Determining the proportion of aggregates, determining the proportion of existing fragments, and oxidation / deamidation A method that includes determining the level.

[36] The method according to

[35] , further comprising determining the biological activity and efficacy of the preparation.

[37] A method for treating human patients suffering from inflammatory bowel disease, wherein the method is Humanized immunoglobulins or their anti-binding specificity for human α4β7 integrin The process includes administering the original bound fragment to human patients suffering from inflammatory bowel disease. Humanized immunoglobulin or its antigen-binding fragments are administered under the following regimen: (a) 300 mg of humanized immunoglobulin or its antigen-binding fragment as an intravenous infusion. One dose, (b) Subsequently, approximately two weeks after the initial dose, 300 mg of humanized immunization was administered intravenously. A second subsequent administration of globulin or its antigen-binding fragment, (c) Subsequently, approximately 6 weeks after the initial dose, 300 mg of humanized immunosorbent immunization was administered intravenously. A third subsequent administration of globulin or its antigen-binding fragment, (d) After the third subsequent administration of the humanized antibody, every 4 weeks or every 8 weeks as needed. 300 mg of humanized immunoglobulin or its antigen-binding fragment administered intravenously (after the fourth administration) It is administered to the patient according to the subsequent administration schedule. The aforementioned administration plan induces clinical response and clinical remission in patients with inflammatory bowel disease; Furthermore, humanized immunoglobulins or antigen-binding fragments may have antigen-binding regions of non-human origin and human origin. It contains at least a portion of the source antibody, and the humanized immunoglobulin or antigen-binding fragment is α4β7 compound It has binding specificity to the combination; the antigen-binding region is CDRs: light chain CDR1: SEQ ID NO: 9 ,CDR2:SEQ ID NO: 10,CDR3:SEQ ID NO: 11;Heavy chain CDR1:SEQ ID NO: 12,C A method including DR2: SEQ ID NO: 13 and CDR3: SEQ ID NO: 14.

[38] Patients who are receiving immunomodulators, tumor necrosis factor α antagonists, or combinations thereof The other lacked an appropriate response, lost the response, or was not recognized as a result of treatment. The method described in

[37] was not suitable.

[39] The method described in

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

[40] The method described in

[39] , where inflammatory bowel disease is ulcerative colitis.

[41] Inflammatory bowel disease is described as moderate to severe active ulcerative colitis.

[39] The method.

[42] The treatment plan is for patients suffering from moderate to severe active ulcerative colitis to achieve mucosal healing. A method for producing

[41]

[43] The treatment plan involves reducing, eliminating, or reducing and eliminating the patient's use of corticosteroids. A method that produces

[37] .

[44] Patients have been treated with at least one corticosteroid for inflammatory bowel disease. A method used by a patient who had previously received treatment.

[37]

[45] Humanized immunoglobulin or its antigen-binding fragments, approximately 1.0 mg / ml to approximately 1.4 The method described in

[37] , administered in the final dose form at a concentration of mg / ml.

[46] Humanized immunoglobulin or its antigen-binding fragment at a concentration of approximately 1.2 mg / ml The method described in

[45] , administered in the final dose form.

[47] Humanized immunoglobulin or its antigen-binding fragment is administered to the patient over a period of approximately 30 minutes. The method described in

[37] .

[48] ​​Humanized immunoglobulin or its antigen-binding fragments are reconstituted from a lyophilized preparation. The method described in

[45] .

[49] Humanized immunoglobulin or its antigen-binding fragments are reconstituted to form a stable liquid formulation. A method for accomplishing

[45]

[50] The administration plan is the ratio of CD4 to CD8 in the cerebrospinal fluid of the patient receiving the treatment. A method that does not change

[37] .

[51] A regimen for the treatment of inflammatory bowel disease, wherein the method involves human α4β7 Humanized immunoglobulin or its antigen-binding fragment having binding specificity to integrins This includes the process of administering the drug to patients suffering from inflammatory bowel disease. Humanized immunoglobulin or its antigen-binding fragments are administered under the following regimen: (a) 300 mg of humanized immunoglobulin or its antigen-binding fragment as an intravenous infusion. One dose, (b) Subsequently, approximately two weeks after the initial dose, 300 mg of humanized immunization was administered intravenously. A second subsequent administration of globulin or its antigen-binding fragment, (c) Subsequently, approximately 6 weeks after the initial dose, 300 mg of humanized immunosorbent immunization was administered intravenously. A third subsequent administration of globulin or its antigen-binding fragment, (d) After the third subsequent administration of the humanized antibody, every 4 weeks or every 8 weeks as needed. 300 mg of humanized immunoglobulin or its antigen-binding fragment administered intravenously (after the fourth administration) Administered to the patient according to the following instructions: The aforementioned administration plan induces clinical response and clinical remission in patients with inflammatory bowel disease; Furthermore, humanized immunoglobulins or antigen-binding fragments may have antigen-binding regions of non-human origin and human origin. It contains at least a portion of the source antibody, and the humanized immunoglobulin or antigen-binding fragment is α4β7 compound It has binding specificity for the combination; the antigen-binding region is the complementarity-determining region (CDRs) shown below. ): Light chain CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11; Heavy Chain CDR1:SEQ ID NO: 12, CDR2:SEQ ID NO: 13, CDR3:SEQ ID NO: 14, Administration plan.

[52] Patients who are receiving at least one immunomodulator, tumor necrosis factor α antagonist or a combination thereof The other lacked an appropriate response, lost the response, or was not recognized as a result of treatment. The administration plan described in

[51] was not suitable.

[53] The administration regimen described in

[51] if the inflammatory bowel disease is Crohn's disease or ulcerative colitis. .

[54] The administration regimen described in

[52] if the inflammatory bowel disease is ulcerative colitis.

[55] Inflammatory bowel disease is described as moderate to severe active ulcerative colitis.

[54] Administration plan.

[56] The treatment plan is for patients suffering from moderate to severe active ulcerative colitis to mucosal healing. The administration plan described in

[55] results in

[55]

[57] The treatment plan involves reducing, eliminating, or reducing and eliminating the patient's use of corticosteroids. The administration plan described in

[51] results in

[51] the following

[58] Patients have been treated with at least one corticosteroid for inflammatory bowel disease. The treatment plan described in

[51] , which the patient had previously received.

[59] Humanized immunoglobulin or its antigen-binding fragments, approximately 1.0 mg / ml to approximately 1.4 The administration regimen described in

[51] is administered in the final dose form at a concentration of mg / ml.

[60] Humanized immunoglobulin or its antigen-binding fragment at a concentration of approximately 1.2 mg / ml The administration regimen described in

[59] is administered in the final dose form.

[61] Humanized immunoglobulin or its antigen-binding fragment is administered to the patient over a period of approximately 30 minutes. The administration plan described in

[51] .

[62] Humanized immunoglobulin or its antigen-binding fragments are reconstituted from a lyophilized preparation. The method described in

[61] .

[63] Humanized immunoglobulin or its antigen-binding fragments are reconstituted to form a stable liquid formulation. The administration plan described in

[61] .

[64] The administration plan is the ratio of CD4 to CD8 in the cerebrospinal fluid of the patient receiving the treatment. The administration regimen described in

[51] does not change the

[51] .

[65] The method of

[0051] , wherein the patient is 65 years of age or older and the patient does not require adjustment of the administration plan.

[66] The administration plan described in

[0051] , wherein the patient is 65 years of age or older and the patient does not require any adjustment to the administration plan.

[67] The preparation according to [1] wherein the molar ratio of free amino acids to antibody is at least 250:1. Agent.

[68] The molar ratio of free amino acids to antibody is at least 250:1

[13] as described above. formulation.

Claims

1. A stable formulation comprising a non-reducing sugar, an anti-α4β7 antibody, and at least one free amino acid. The formulation contains a mixture, and the formulation is in solid form, with a molar ratio of non-reducing sugar to anti-α4β7 antibody (moles: A stable formulation with a mole ratio exceeding 600:

1.

2. The formulation according to claim 1, further comprising a buffering agent.

3. The aforementioned non-reducing sugars include mannitol, sorbitol, sucrose, trehalose, and the like. A formulation according to claim 1, selected from a group consisting of combinations.

4. The aforementioned free amino acids are histidine, alanine, arginine, glycine, and glutamic acid. The formulation according to claim 1, selected from the group consisting of and combinations thereof.

5. The formulation according to claim 1, further comprising a surfactant.

6. The surfactant is polysorbate 20, polysorbate 80, poloxamer and the The formulation according to claim 5, selected from the group consisting of combinations thereof.

7. The formulation is freeze-dried, and before freeze-drying, at least about 5% to about 10% of anti-α4β7 antibodies The formulation according to claim 1, comprising:

8. The formulation according to claim 1, comprising at least about 6% anti-α4β7 antibody before lyophilization. formulation.

9. The formulation comprises free amino acids in a concentration between approximately 100 mM and approximately 175 mM, as described in claim 1. A formulation of [the substance].

10. The formulation according to claim 1, wherein the formulation is a liquid formulation.

11. The formulation according to claim 1, wherein the formulation is a dried formulation.

12. The formulation according to claim 1, wherein the formulation is stable for at least 3 months at 40°C and 75% RH. 。

13. A stable liquid formulation comprising a non-reducing sugar, an anti-α4β7 antibody, and at least one other in aqueous solution. It contains free amino acids, and the molar ratio (moles:moles) of non-reducing sugar to anti-α4β7 antibody is 600:1 A liquid formulation that is more stable than [presumably referring to a specific type of formulation].

14. The liquid formulation according to claim 13, further comprising a buffering agent.

15. The aforementioned non-reducing sugars include mannitol, sorbitol, sucrose, trehalose, and the like. A liquid formulation according to claim 13, selected from the group consisting of combinations.

16. The aforementioned free amino acids are histidine, alanine, arginine, glycine, and glutamic acid. A liquid formulation according to claim 13, selected from the group consisting of and combinations thereof.

17. The liquid formulation according to claim 13, further comprising a surfactant.

18. The surfactant is polysorbate 20, polysorbate 80, poloxamer and the A liquid formulation according to claim 17, selected from the group consisting of combinations thereof.

19. The liquid formulation according to claim 13, wherein the formulation has a pH between approximately 5.5 and approximately 7.

5.

20. The liquid formulation according to claim 13, wherein the formulation has a pH between approximately 6.0 and approximately 6.

5.

21. The formulation contains at least about 60 mg / ml to about 80 mg / ml of anti-α4β7 antibody The liquid formulation according to claim 13.

22. The formulation according to claim 13, comprising at least about 60 mg / ml of anti-α4β7 antibody. Liquid formulation.

23. A stable formulation containing at least approximately 60 mg / ml to approximately 80 mg / ml of anti-α4β7 anti- The preparation contains a body, a buffering agent, and at least about 10% (w / w) of sugar, and the preparation is a liquid preparation. A standard formulation.

24. The formulation according to claim 23, wherein the buffering agent is a histidine buffer.

25. The formulation according to claim 23, wherein the sugar is sucrose.

26. A stable formulation comprising at least about 60 mg / ml of anti-α4β7 antibody and at least about 1 A stable formulation containing 0% (w / w) non-reducing sugars, wherein the formulation is freeze-dried.

27. The formulation according to claim 26, wherein the formulation further comprises polysorbate 80.

28. The formulation according to claim 26, wherein the non-reducing sugar is sucrose.

29. A stable formulation containing non-reducing sugar, anti-α4β7 antibody, histidine, arginine, and polysol The formulation contains a mixture of β-80, and the formulation is in solid form, and is a mixture of non-reducing sugar and anti-α4β7 antibody. A stable formulation with a mole-to-mol ratio (moles:moles) exceeding 600:

1.

30. The formulation according to any one of claims 13 to 25, further comprising a metal chelating agent.

31. The formulation according to any one of claims 13 to 25, further comprising an antioxidant.

32. The formulation according to any one of claims 1 to 31, wherein the antibody is vedolizumab.

33. A method for producing a pharmaceutical product according to any one of claims 1 to 32, wherein the method is primary A method that includes maintaining a product temperature below the decay temperature during drying.

34. The method according to claim 33, further comprising an annealing step.

35. A method for determining the quality of a freeze-dried preparation, which involves inspecting the preparation for its appearance. This involves determining the time of reconstruction, determining the water content, and the existence of aggregates. Determining the ratios, determining the ratio of the existing fragments, and the oxidation / deamidation levels. A method that includes determining the answer.

36. The method according to claim 35, further comprising determining the biological activity and efficacy of the formulation.

37. A method for treating human patients suffering from inflammatory bowel disease, wherein the method is Humanized immunoglobulin having binding specificity to human α4β7 integrin or its anti- The process includes administering the original bound fragment to human patients suffering from inflammatory bowel disease. Humanized immunoglobulin or its antigen-binding fragments are administered under the following regimen: (a) Initial administration of 300 mg of humanized immunoglobulin or its antigen-binding fragment as intravenous infusion One dose, (b) Subsequently, approximately two weeks after the initial dose, 300 mg of humanized immunosorbent benzoate was administered intravenously. A second subsequent administration of globulin or its antigen-binding fragment, (c) Subsequently, approximately 6 weeks after the initial dose, 300 mg of humanized immunosorbent was administered intravenously. A third subsequent administration of globulin or its antigen-binding fragment, (d) After the third subsequent administration of the humanized antibody, every 4 weeks or every 8 weeks as needed 300 mg of humanized immunoglobulin or its antigen-binding fragment administered intravenously (after the fourth administration) It is administered to the patient according to the subsequent administration schedule. The aforementioned administration plan induces clinical response and clinical remission in patients with inflammatory bowel disease; Furthermore, humanized immunoglobulins or antigen-binding fragments may have antigen-binding regions of non-human origin and human origin. It contains at least a portion of the source antibody, and the humanized immunoglobulin or antigen-binding fragment is α4β7 compound It has binding specificity to the combined form; the antigen-binding region is CDRs: light chain CDR1: SEQ ID NO: 9 CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11; Heavy chain CDR1: SEQ ID NO: 12, C A method including DR2: SEQ ID NO: 13, CDR3: SEQ ID NO:

14.

38. The patient is receiving at least one immunomodulator, tumor necrosis factor alpha antagonist, or a combination thereof. They lacked an appropriate response to, lost the ability to respond to, or were not tolerable to treatment as a result. The method described in claim 37, which was not present.

39. The method according to claim 37, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

40. The method according to claim 39, wherein the inflammatory bowel disease is ulcerative colitis.

41. The person according to claim 39, wherein the inflammatory bowel disease is moderate to severe active ulcerative colitis. Law.

42. The administration plan induces mucosal healing in patients suffering from moderate to severe active ulcerative colitis. The method according to claim 41.

43. The administration plan results in the reduction, elimination, or reduction and elimination of the patient's use of corticosteroids. The method according to claim 37.

44. The patient has been treated with at least one corticosteroid for inflammatory bowel disease. The method described in claim 37, which was previously received.

45. Humanized immunoglobulin or its antigen-binding fragments are present in a concentration of approximately 1.0 mg / ml to approximately 1.4 mg / ml. The method according to claim 37, wherein the final dose is administered at a concentration of ml.

46. Humanized immunoglobulin or its antigen-binding fragment was administered at a final dose of approximately 1.2 mg / ml. The method according to claim 45, administered in a given form.

47. Claim that humanized immunoglobulin or its antigen-binding fragment is administered to a patient within approximately 30 minutes. Method 37.

48. Humanized immunoglobulin or its antigen-binding fragments are reconstituted from lyophilized preparations. The method described in item 45.

49. Humanized immunoglobulin or its antigen-binding fragments are reconstituted to form a stable liquid formulation. The method according to claim 45.

50. The administration plan alters the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving the aforementioned treatment. The method according to claim 37, which prevents such occurrence.

51. A treatment plan for inflammatory bowel disease, wherein the method involves human α4β7 nitrate Humanized immunoglobulin or its antigen-binding fragment having binding specificity to glycin is used in inflammation. This includes the process of administering the drug to patients suffering from colorectal disease. Humanized immunoglobulin or its antigen-binding fragments are administered under the following regimen: (a) Initial administration of 300 mg of humanized immunoglobulin or its antigen-binding fragment as intravenous infusion One dose, (b) Subsequently, approximately two weeks after the initial dose, 300 mg of humanized immunosorbent benzoate was administered intravenously. A second subsequent administration of globulin or its antigen-binding fragment, (c) Subsequently, approximately 6 weeks after the initial dose, 300 mg of humanized immunosorbent was administered intravenously. A third subsequent administration of globulin or its antigen-binding fragment, (d) After the third subsequent administration of the humanized antibody, every 4 weeks or every 8 weeks as needed 300 mg of humanized immunoglobulin or its antigen-binding fragment administered intravenously (after the fourth administration) Subsequent administration; administered to the patient according to the instructions. The aforementioned administration plan induces clinical response and clinical remission in patients with inflammatory bowel disease; Furthermore, humanized immunoglobulins or antigen-binding fragments may have antigen-binding regions of non-human origin and human origin. It contains at least a portion of the source antibody, and the humanized immunoglobulin or antigen-binding fragment is α4β7 compound It has binding specificity for fusion; the antigen-binding region is the complementarity-determining region (CDRs) shown below. ): Light chain CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11; Heavy Chain CDR1: containing SEQ ID NO: 12, CDR2: containing SEQ ID NO: 13, CDR3: containing SEQ ID NO: 14, Administration plan.

52. The patient is receiving at least one immunomodulator, tumor necrosis factor alpha antagonist, or a combination thereof. They lacked an appropriate response to, lost the ability to respond to, or were not tolerable to treatment as a result. The administration plan described in claim 51 was not present.

53. The administration plan according to claim 51, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

54. The administration plan according to claim 52, wherein the inflammatory bowel disease is ulcerative colitis.

55. The present invention according to claim 54, wherein the inflammatory bowel disease is moderate to severe active ulcerative colitis. Given the plan.

56. The administration plan induces mucosal healing in patients suffering from moderate to severe active ulcerative colitis. The administration plan according to claim 55.

57. The administration plan results in the reduction, elimination, or reduction and elimination of the patient's use of corticosteroids. The administration plan according to claim 51.

58. The patient has been treated with at least one corticosteroid for inflammatory bowel disease. The administration plan described in claim 51 that was previously received.

59. Humanized immunoglobulin or its antigen-binding fragments are present in a concentration of approximately 1.0 mg / ml to approximately 1.4 mg / ml. The administration plan according to claim 51, wherein the final dose is administered at a concentration of ml.

60. Humanized immunoglobulin or its antigen-binding fragment was administered at a final dose of approximately 1.2 mg / ml. The administration plan according to claim 59, which is administered in a given form.

61. Claim that humanized immunoglobulin or its antigen-binding fragment is administered to a patient within approximately 30 minutes. The administration plan described in 51.

62. Humanized immunoglobulin or its antigen-binding fragments are reconstituted from lyophilized preparations. The method described in item 61.

63. Humanized immunoglobulin or its antigen-binding fragments are reconstituted to form a stable liquid formulation. The administration plan according to claim 61.

64. The administration plan alters the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving the aforementioned treatment. The administration plan described in claim 51, which prevents the administration of the drug.

65. Claim 51: The patient is 65 years of age or older, and the patient does not require adjustment of the administration plan. Methods used.

66. Claim 51: The patient is 65 years of age or older, and the patient does not require adjustment of the administration plan. The administration plan described below.

67. The formulation according to claim 1, wherein the molar ratio of free amino acids to antibody is at least 250:

1.

68. The formulation according to claim 13, wherein the molar ratio of free amino acids to antibody is at least 250:

1. 。

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