stable formulation, its method of preparation and its use, as well as the use of a mixture of a non-

专利摘要:
STABLE FORMULATION, ITS METHOD OF PREPARATION AND USE, AS WELL AS USE OF A MIXTURE OF A NON-REDUCING SUGAR, AN ANTI-(Alpha)4(Beta)7 ANTIBODY AND AT LEAST ONE FREE AMINO ACID. Antibody formulations are described comprising a mixture of a non-reducing sugar, an anti- (Alpha)4(Beta)7 antibody and at least one free amino acid. The disclosed formulations have improved stability, reduced aggregate formation and may delay degradation of the anti-(Alpha)4(Beta)7 antibody therein or exhibit any combinations thereof. The present invention further provides a safe dosing regimen of these antibody formulations which is easy to follow and which results in a therapeutically effective amount of the anti-(Alpha)4(Beta)7 antibody in vivo.
公开号:BR112013028424A2
申请号:R112013028424-2
申请日:2012-05-02
公开日:2021-05-04
发明作者:Willow Diluzio；Nobel T. Truong；Csanad M. Varga；Vaithianathan Palaniappan；Jason Brown；Irving H. Fox；Catherine Scholz
申请人:Millennium Pharmaceuticals, Inc.；
IPC主号:

专利说明:

STABLE FORMULATION, ITS METHOD OF PREPARATION AND USE, AS WELL AS USE OF A MIXTURE OF A NON-REDUCING SUGAR, AN ANTI-α4β7 ANTIBODY AND AT LEAST ONE FREE AMINO ACID 5 RELATED ORDERS This application claims the benefit of provisional application US 61/585,859, filed January 12, 2012, and provisional application US 61/550,545 filed October 24, 2011, and provisional application US 61/481,533 filed May 2, 2011. The full contents of the foregoing applications are hereby incorporated for reference.
SEQUENCE LISTING The present application contains a Sequence Listing which has been presented in ASCII format via EFS-Web and is incorporated by reference in its entirety. Said ASCII copy, created on April 30, 2012, is named 92596615.txt and is 17,024 bytes in size.
BACKGROUND OF THE INVENTION Advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA techniques. Due to the fact that proteins are larger and more complex than traditional organic and inorganic drugs (that is, they have several functional groups in addition to complex three-dimensional structures), the formulation of such proteins has special problems. For a protein to remain biologically active, a formulation must preserve the conformational integrity of at least a core sequence of the protein's amino acids, while at the same time protecting the protein's various functional groups from degradation. Proteins can suffer from a lack of stability and monoclonal and polyclonal antibodies, in particular, can be relatively unstable (See, for example, Wang et al., J. Pharm Sci. 96:1-26 (2007)). A large number of formulation options are available and no single approach or system is available for all proteins. Several factors to consider have been reported (See, for example, Wang et al.). A number of features can affect the stability of a protein. In fact, even in the case of purified antibodies, antibody structures can be heterogeneous, which further complicates the formulation of such systems. Furthermore, excipients included in antibody formulations preferably minimize any potential immune response. In the case of antibodies, preservation of conformational integrity is even more important. Degradation pathways for proteins may involve chemical instability (ie, any process that involves modification of the protein by bond formation or cleavage resulting in a novel chemical entity) or physical instability (ie, alterations to the higher-order structure of the protein) . Chemical instability is manifested in, for example, deamidation, isomerization, hydrolysis, oxidation, fragmentation, beta glycan elimination, or disulfide exchange.
Chemical instability can result from denaturation, aggregation, precipitation or adsorption, for example.
The four most common protein degradation pathways are protein breakdown, aggregation, deamidation, and oxidation.
Consequences of therapeutic protein chemical or physical instability include a reduction in the effective dose administered, reduced safety of therapy due to, for example, irritation or immune reactivity, and, more often, manufacturing due to short half-life.
Freeze drying is a technique commonly used to preserve proteins; lyophilization serves to remove water from the protein preparation of interest.
Freeze-drying, or lyophilization, is a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum.
Excipients can be included in the pre-lyophilized formulation to stabilize proteins during the lyophilization process and/or to improve the stability of the lyophilized protein formulation (Pikal M., Biopharm. 3(9)26-30 (1990) and Arakawa ¢at al.
Pharm.
Res. 8(3):285-291 (199j)). Various publications have generally disclosed various methods for treating inflammatory bowel disease and provided dosing schedules for administering agents designed to treat inflammatory bowel disease.
For example, WO 96/24673 discloses mucosal vascular addressins and diseases associated with leukocyte recruitment to the gastrointestinal tract as a result of leukocyte binding to cells expressing MAdCAM.
US 2005/0095238 describes methods for treating a disease associated with leukocyte infiltration of mucosal tissue and administering to a human an effective amount of a human or humanized immunoglobulin or antigen-binding fragment having binding specificity for α4,37 integrin.
US 2005/0095238 further describes various doses (eg 0.15, about 0.5, about 1.0,
about 1.5 or about 2.0 mg of immunoglobulin or fat per kg of body weight) and various intervals between doses (7, 14, 21, 28, or 30 days). However, the aforementioned patents and publications do not disclose the specific anti-α417 antibody formulations or the specific doses and dose regimens claimed herein.
Importantly, the aforementioned patents do not disclose formulations, doses, and dose ranges that provide the methods of treatment (supported by clinical study data) described and claimed herein. The antibody formulations of the present invention may be useful to inhibit leukocyte binding to cells expressing MAdCAM and, therefore, aid in the treatment of inflammatory bowel disease in patients. There is, therefore, an urgent need to discover appropriate dosages and dosage schedules of these compounds, and to develop formulations, preferably intravenous formulations, that generate stable, therapeutically effective blood levels of the antibody formulations for an extended period of time in a Stable and convenient way.
SUMMARY OF THE INVENTION The invention relates to identifying a non-reducing sugar and at least one amino acid as useful excipients for formulating anti-α437 antibody formulations whose instability makes them susceptible to deamidation, oxidation, isomerization and/or aggregation. Formitlation improves stability, reduces aggregate formation and retards antibody degradation therein. Thus, in a first aspect, the invention relates to a formulation comprising a mixture of a non-reducing sugar, anti-α4j37 antibody, and at least one free amino acid and the molar ratio of non-reducing sugar to anti-α4j37 antibody (mol :mol) is greater than 600:1. The formulation can be a liquid formulation or a dry formulation (for example, lyophilized). The formulation may further contain a buffering agent. In some embodiments, the non-reducing sugar is mannitol, sorbitol, sucrose, treabse, or any combination thereof. In some embodiments, the free amino acid of the formulation is histidine, alanine, arginine, glycine, glutaniic acid, or any combination thereof. The formulation can comprise from about 50 mM to about 175 nM of the free amino acid. The formulation can comprise between about 100 mM and about 175 mM of the free amino acid. The molar ratio of free amino acid to molar ratio of antibody is at least 250:1.
The formulation may also contain a surfactant. The surfactant can be polysorbate 20, polysorbate 80, a poloxamer, or any combination of the same.
In some aspects, the formulation can minimize the immunogenicity of the anti- 4B7 antibody.
The formulation, for example, in the dried state, can be stable for at least three months at 40'C, 75% relative humidity (RH). In another aspect, formulation C is lyophilized and comprises at least about 5% to about ICP/anti-o437 antibody prior to lyophilization.
The formulation may contain at least about 6.4% anti-a4B7 antibody prior to lyophilization.
The formulation can be reconstituted from a lyophilized formulation (e.g., reconstituted to comprise a stable liquid formulation). In another aspect, the invention relates to a stable formulation comprising a mixture of a non-reducing sugar, an anti-u4B7 antibody and at least one free amino acid, and the molar ratio of non-reducing sugar to anti-a4f17 antibody (mol: mol) is greater than 10 than 600:1 and the ratio of free amino acid to anti-α437 antibody (lnol:no]) is greater than 250:1. In another aspect, the invention relates to a stable liquid formulation comprising in aqueous solution with a non-reducing sugar, an anti-α437 antibody, and at least one free amino acid, wherein the molar ratio of non-reducing sugar to anti-IS antibody is 0t4B7 (mol:mol) is greater than 600:1.
In yet another aspect, the invention relates to a liquid formulation comprising at least about 40 mg/ml to about 80 mg/ml anti-a4B7 antibody, at least about 50-175 mM of one or more amino acids, and at least about 6% to at least about JO'/q (w/v) sugar.
The liquid formulation may further contain a buffering agent.
In some embodiments the liquid formulation further comprises a metal chelator.
In some embodiments, the liquid formulation still contains an antioxidant.
In another aspect, the invention relates to a liquid formulation comprising at least about 60 mg/ml anti-u7, at least about 10% (w/v) non-reducing sugar, and at least about 125 mM one or more free amino acids.
In another aspect, the invention relates to a liquid formulation comprising at least about 60 mg/ml anti-u437 antibody, at least about 10-4 (w/v) non-reducing sugar, and at least about 175 mM of one or more free amino acids In yet another aspect, the invention relates to a dry, e.g., lyophilized, formulation comprising a mixture of a non-reducing sugar, an anti-α437 antibody, histidine, arginine, and polysorbate 80, in that the formulation is in solid form, and the molar ratio of non-reducing sugar to anti-cL4B7 antibody (mol:mol) is greater than 600:1. In yet another aspect, the invention relates to a lyophilized formulation
T"" 5/91 comprising a mixture of a non-reducing sugar, an anti-44B7 antibody, histidine, arginine, and polysorbate 80. In this aspect, the molar ratio of non-reducing sugar to anti-α4j37 antibody (mol:mol) is greater than 600:1. In addition, the molar ratio of arginine to anti-α437 antibody (mol:mol) in the formulation is greater than 250:1. In another aspect, the invention relates to a method for preparing a formulation described herein, comprising maintaining the temperature of the product below the collapse temperature during primary drying.
The method can also contain an annealing step.
In one aspect, the invention relates to a method of treating a human patient suffering from inflammatory bowel disease, the method comprising the steps of administering to a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or fragment of antigen binding thereof containing specificity for human q4B7 integrin, wherein the immunized immunoglobulin or antigen-binding fragment comprises an antigen-binding region of non-human origin and at least a part of an antibody of human origin, wherein the immunoglobulin The immunized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the following dosing regimen: (a) an initial dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion; (b) followed by a second subsequent dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion at approximately two weeks 20 after the initial dose; (c) followed by a third subsequent dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion at about six weeks after the initial dose; (d) followed by a fourth subsequent 300 µg dose of the immunoglobulin or mesnia antigen binding fragment with an intravenous infusion every four weeks after the third subsequent dose of humanized antibody, as needed; wherein the dosage regimen induces a clinical response and/or clinical remission in the patient's inflammatory bowel disease; and further wherein the immunized immunoglobulin or antigen-binding fragment has binding specificity for the a4B7 complex, wherein the antigen-binding region comprises three complementarity-determining regions (CDRI, CDR2, 30 and CDR3) of a variable region of light chain and three complementarity determining regions (CDRI, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set out below: light chain: CDRI SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:ll; heavy chain: CDRI SEQ ID NO:12, CDR2 SEQ
" ID NO:13, CDR3 SEQ ID NO:14.
In another aspect, the invention relates to a dosage regimen for the therapeutic treatment of inflammatory bowel disease, wherein the dosage regimen comprises the step of: administering to a patient suffering from inflammatory bowel disease, the immunized immunoglobulin or An antigen-binding segment thereof containing specificity for human 44B7 integrin, wherein the immunized immunoglobulin or antigen-binding segment comprises an antigen-binding region of non-human origin and at least a portion of an antibody of human origin, wherein the immunized immunoglobulin or antigen-binding fragment thereof is administered to the patient according to the following dosing regimen: (a) an initial dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion; (b) followed by a second subsequent dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion within about two weeks after the initial dose; (c) followed by a subsequent third dose of 300 mg of the immunized immunoglobulin or antigen-binding fragment thereof with an intravenous infusion about six weeks after the initial dose; (d) followed by a fourth and subsequent 300 mg doses of the immunized immunoglobulin or antigen-binding fragment thereof as an intravenous infusion every four weeks or every eight weeks after the subsequent third dose of the humanized antibody, as needed; wherein the dosing regimen induces a clinical response and/or clinical remission of the patient's ir]testial inflammatory disease; and airida wherein the humanized immunoglobulin or antigen-binding fragment has binding specificity for the ct4j37 complex, wherein the antigen-binding region comprises three complementarity determining regions (CDRI, CDR2, and CDR3) of a chain variable region light and three complementarity determining regions (CDRI, CDR2, and CDR3) of a heavy chain variable region of the amino acid sequence set forth herein below: light chain: CDRI SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:ll; heavy chain: CI)R1 SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3 SEQ ID NO:14.
In some aspects, the method of treatment with the anti-a4B7 antibody formulation, dose or dosing regimen can minimize the immunogenicity of the anti-(L4B7 antibody.)
The patient may have had an adequate lack of response with 7/91, loss of response to ar"""""" or was intolerant of treatment with at least one of an immunomodulator, a tumor necrosis factor alpha (TNF-α) antagonist ) or combinations thereof.
Inflammatory bowel disease can be Crohn's disease or ulcerative colitis. Innamatory bowel disease can be moderated to severely active ulcerative colitis.
5 The dosing regimen can result in mucosal healing in patients suffering from moderately to severely active ulcerative colitis.
The patient may have previously received treatment with at least one corticosteroid for inflammatory bowel disease. The dosing regimen may result in a reduction, elimination or reduction and elimination of conicosteroid use by the patient.
In some embodiments, the humanized immunoglobulin or antigen-binding fragment thereof is administered in a final dosage form at a concentration of from about 1.0 mg/ml to about 1.4 mg/ml. Humanized immunoglobulin or antigen-binding fragment thereof can be administered in a final dosage form of about 1.2 mg/ml. The humanized immunoglobulin or L5 antigen-binding fragment can be administered to the patient in about 30 minutes. The immunized immunoglobulin or antigen-binding fragment thereof can be reconstituted from a lyophilized formulation.
The immunized immunoglobulin or antigen-binding fragment thereof can be reconstituted to comprise a stable liquid formulation.
20 In some respects, the dosing regimen does not alter the ratio of CD4 to CD8 in the cerebrospinal fluid of patients receiving such treatment. The patient can be a person 65 years of age or older and does not require any adjustment to the dnse regimen.
BRIEF DESCRIPTION OF THE DRAWINGS 25 FIG. 1 is an illustration of a nucleotide sequence (SEQ ID NO:1) encoding the heavy chain of a humanized anti-cL4B7 immunoglobulin, and the deduced heavy chain amino acid sequence (SEQ ID NO:2). The nucleotide sequence contains cloning sites (low box), Kozak sequence (high box, nucleotides 18-23 of SEQ ID NO:I) and leader sequence (low box, nucleotides 24-86 of SEQ ID NO:1) in 30 5' end of heavy chain. The open reading frame of the nucleotide sequence is nucleotides 24-1433 of SEQ JD NO:1.
The FJG. 2 is an illustration of a nucleotide sequence (SEQ ID NO:3) encoding the light chain of a humanized immunoglobulin referred to herein as vedolizumab, and the resulting amino acid sequence (SEQ TD NO:4) of the light chain. The nucleotide sequence contains cloning sites (low box), Kozak sequence (high box, nucleotides 18-23 of SEQ ID NO:3) and leader sequence (low box, nucleotide 24-80 of SEQ ID NO:3) at the end 5' of the heavy chain.
The open reading region of the nucleotide sequence is nucleotides 24-737 of SEQ ID NO:3. FIG. 3 is an alignment of the amino acid sequences of (A) the mature humanized light chain (amino acids 20-238 of SEQ ID NO:4) of a humanized immunoglobulin referred to herein as vedolizumab and (B) the mature humanized light chain of a humanized immunoglobulin referenced herein as LDP-02 (SEQ ID NO:5). (With regard to LDP-02, vcr, WO 98/06248 and Feagan et al., N.
Eng, ./. Med. 352:2499-2507 (2005)) - Feagan et al. describes a clinical study of LDP-02, but in the article they refer to LDP-02 as MLN02.) The alignment illustrates that the amino acid sequences of the levcs chains of vedolizumab and J.DP-02 differ at positions 114 and 11S of the chains ripe light.
FIG. 4 is an amino acid sequence alignment of (A) a generic human kappa light chain constant region (SEQ ID NO:6) and (B) a generic murine kappa light chain constant region (SEQ ID NO:7) . Amino acid residues Thr and Val (which are present at positions 114 and 115 of the mature vedolizumab light chain (amino acids 133 and 134 of SEQ ID NO:4)) are present in the constant retention of the human kappa chain, while the amino acid residues Ala and Asp (which are present at positions 1-4 and 115 of the mature LDP-02 light chain (SEQ ID NO:5)) are present in the constant reaction of the mouse kappa light chain.
FIG. 5 is a vector map of pLKTOK38D (further referenced as pTOK3XMLN02TV), which encodes the humanized heavy chain and the humanized light chain of MLN02, and is suitable for production in CHO cells. (See, US patent application publication 2004/0033561 A1 which revs pLKTOK38. pLKTOK38D is a variant of pLKTOK38 in which restriction sites on the map flank the light chain variable region encoding sequence.) FIG. 6 A shows the predictive models for change in percent monomer, change in percent aggregate, and change in percent major isoform of the lyophilized anti-cy437 formulation.
The models are based on statistical analyzes of the data presented in Example 1. The middle row shows the results of predictive models ¢ the outer rows show a 9S°/o confidence limit for predictive models.
FIG. 6B shows alternative models based on statistical analysis of 40°C data from r" 9/91" Tables 1-3 when the input factors are pH, sugar:protein motar ratio, c arginine:protein molar ratio. The center line shows the results of predictive models and the outer lines show a confidence limit of 9S'/o for predictive models. FIG. 7 shows the amino acid sequences of (A) human mature GM607'CL antibody 5 kappa light chain variable region and (B) 21/28'CI heavy chain variable region. huniana. FIG. 8 is a graph showing that solids and loading affect drying time (numbers in rows represent number of minutes of drying time) - FIG 9 is a graph showing vedolizumab did not delay the onset of 10 clinical symptoms of autoimmune encephalomyelitis (EAE) compared to placebo control. Natalizumab significantly (p<0.05) delayed the onset of clinical symptoms of EAE compared to pIacebo control.
DETAILED DESCRIPTION OF THE INVENTION The invention relates to formulations comprising anti-u4B7 antibodies. The 15 formulations can be mixtures comprising non-reducing sugar, anti-α4j37 antibody and one or more free amino acids, the molar ratio of non-reducing sugar to anti-α4fl17 antibody is greater than 600 moks of non-reducing sugar:1 mol of anti-antibody -ct4[37. The formulations can be in solid or liquid form. Definitions 20 The term "pharmaceutical formulation" refers to a preparation which contains an anti-α437 antibody in such a way as to allow the biological activity to be effective, and which does not contain additional components which are unacceptably toxic to a subject to which The Formulation Could Be Administered A "stable" formulation is one in which the antibody therein substantially retains its physical stability and/or its chemical stability and/or its biological activity in storage. In one aspect, the formulation substantially retains its physical and chemical stability, as well as its biological activity on storage. The storage period is generally selected based on the shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, in Peptide and Pro/('in Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (] 991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example A "deamidated" monoclonal antibody is one in which one or more asparagine or glutamine residues has been derived therefrom. , for example, to an aspartic acid or an isoaspartic acid.
An antibody that is "susceptible to deainidation" is one comprising one or more residues that have been shown to be prone to deamidate. 5 An antibody that is "susceptible to oxidation" is an antibody comprising one or more residues that have been shown to be prone to oxidation.
An antibody that is "susceptible to aggregation" is one that has been shown to aggregate with other antibody molecules, especially on freezing, heating, drying, reconstitution, and/or agitation. 10 An antibody that is "susceptible to fragmentation" is one that has been shown to be cleaved into two or more fragments, for example, in a hinge region thereof.
By "reduce dearnidation, oxidation, aggregation, or fractionation" is meant to prevent or reduce (e.g., to 80°4, 60%, 50%, 40'4, 30%, 20% or 10% of) the amount of decamidation, aggregation, or fraymentation relative to the monoclonal antibody formulated in a different pII or a different buffer.
An "aggregate", "SEC aggregate", or "soluble aggregate" is more than one and less than or equal to ten antibody proteins and/or fragments associated together or by covalent, ionic or hydrophobic interactions to form a protein body bigger.
An "insoluble aggregate" or "particle" is larger than ten antibody proteins and/or associated fragments or by covalent, ionic, or hydrophobic interactions to form a larger protein body.
As used herein, "biological activity" of a monoclonal antibody refers to the antibody's ability to bind antigen and result in a measurable biological response that can be measured in vitro or in vivo.
Said activity can be antagonistic or agonistic.
The cell surface molecule, 'h4[37 integrin," or "ct4|37," is a heterodimer of one q4 chain (CD49D, ITGA4) and one p7 chain (itgb7). alternative integrin, to form mlh or aEB7. Human q4 and p7 genes (Gen8ank (National Center for Biotechnology Information, Belhesda, MD) RefSeq accession numbers NM _ 000885 and NM _ 000889, respectively) are expressed by B and T lymphocytes, particularly CD4+ Memory Lymphocytes Typical of many integrins, a4B7 can exist in a resting or activated state.
Ligands for a4B7 include vascular cell adhesion molecule (VCAM), fibronectin and mucosal addressin
(MAdCAM, eg MAdCAM-1).
As used herein, a human immunoglobulin or antigen-binding fragment thereof that has "binding specificity for the cc4f17 complex" binds to q4B7 but not to c4B] or áEB7.
5 As used here, an "isotonic" formulation has substantially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freeze-type osmometer, for example.
As used herein, "buffering agent" refers to a buffer that resists changes in pH through the action of its acid-base conjugate components. The tanning agent can be present in a liquid or solid formulation of the invention. The tanning agent adjusts the pH of the formulation to about 5.0 to about 7.5, to about 5.5 to about 7.5, to about 6.0 to about 6.5, or to about a pH of about 6.3. In one aspect, examples of buffering agents that will control the pH in the 5.0 to 7.5 range include acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, cacodylate, 2-µ-morpho]ino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxynitro]]methane (81s-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and other organic acid buffers. In another aspect, the buffering agent here is histidine or citrate.
A "histidine buffer" is a buffer comprising histidine groups. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate solutions. The histidine buffer or histidine-HCl buffer has a pH of between about pH 5.5 to 6.5, between about pH 6.1 to about 6.5, or about pH 6.3.
A "saccharide" here is a compound having a general formula (CH2O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. In one aspect, examples of saccharides herein include glucose, sucrose, trehalose, lactose, phmtose, maltose, dextran, eryritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, mekzitose, raffinose, manotriosis, nythiose, maltose [altu]ose, glucitol, maltite], lactitol, isomaltulose, and the like. A saccharide can be a lyoprotectant. In another aspect, the saccharide here is a non-reducing disaccharide such as sucrose. A "surfactant" here refers to an agent that reduces the surface tension of a liquid. The surfactant can be a non-ionic surfactant. In one aspect examples of
T" 12/91 surfactants herein include polysorbate (polyoxyethylene sorbitan monolaurate, e.g., polysorbate 20 and polysorbate 80); TRITON (t-Octylphenoxypolyethoxyethanol, nonionic detergent, Union Carbide subsidiary of Dow Chemical Co., Midland MI); dodecyl sulfate of dowland MI; sodium (SDS); sodium lauryl sulfate; octyl sodium glycoside; lauryl-, myristyl-, linoleyl-, or 5-stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (for example, lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; methyl disodium; sorbitan monopalmitate; and the MONAQUAT 10 series (Mona Industries, Inc., Paterson, Nj); polyethylene glycol (PEG), polypropylene glycol (PPG), and polyoxyethylene and polyoxypropylene glycol copolymers (e.g., Pluronics/Po sander, PF68 etc); etc. In another aspect, the surfactant is polysorbate 80. The term "antibody" herein is used in the broadest sense and specifically covers the complete monoclonal antibodies, immunoglobulins, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two full-length antibodies, for example, each to a different antigen or epitope, and individual antigen-binding fragments, including dAbs, scFv, Fab, F(ab)'2, Fab', including human and humanized antibodies from non-species human and recombinant antigen-binding forms such as monobodies and diabodies.
Amounts and molar ratios of anti-C437 antibody to other excipients described herein are calculated assuming an approximate molecular weight of about 150,000 daltons for the antibody. The actual molecular weight of the antibody may differ from 150,000 daltons, depending on the amino acid composition or post-translational modification, for example, as dependent on the cell lineage used to express the antibody. The actual molecular weight of the antibody may be +/- 5° /) of 150,000 daltons. The term "human antibody" includes an antibody that has a sequence that is derived from a human germline immunoglobulin sequence, such as an antibody derived from transgenic mice containing human immunoglobulin genes (eg, genetically modified XENOMOUSE mice (Abgenix, Fremont, 30 CA), HUMAB-MOUSE ®, transchromosomal mice KIRIN TC MOUSE", KMMOUSE® (M EDAREX, Princeton, NJ)), human phage display libraries, human Myeloma cells, or human B cells. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants which may arise during monoclonal antibody production, said variants generally being present in smaller amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring 10 production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by recombinant DNA methods (see , for example, US patent 4,816,567). "Monoclonal antibodies" can further be isolated from phage antibody libraries using the techniques described in Cyackson et al., Ncuure, 352:624-628 (1991) and Marks ('/ al., ./. Mol. Biol., 222:581-597 (1991), for example Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or which belongs to a particular class or subclass of antibody or, while the remainder of the chains is identical with or homologous to the corresponding sequences in antibodies derived from other species or which belong to another class or subclass of antibodies, as well as fragments of such antibodies, however that present the desired biologic activity (US patent
4.8 16,567; and Morrison et al., Proc. Na/l. Academic Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences. "Angen binding fragments" of a humanized immunoglobulin prepared in the formulation of the invention comprise at least the heavy and/or light chain variable regions of an anti-«²4B7 antibody. For example, an antigen-binding kagment of vedo]izun]ab¢ comprises amino acid residues 20-131 of the humanized light chain sequence of SEQ ID NO:4. Examples of such antigen-binding flags include Fab fragments, Fab' fragments, scFv and F(ab')2 fragments of a humanized immunoglobulin known in the art. Antigen-binding fragments of a humanized immunoglobulin of the invention can be produced by enzymatic cleavage or by recombinant techniques. For example, cleavage by papain or pepsin can be used to generate Fab or F(ab')2 fragments, respectively. Antibodies can further be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a recombinant construct that encodes the heavy chain of an F(ab')z fragment can be designed to include DNA sequences that encode the CH domain, and heavy chain hinge region. In one aspect, antigen-binding fragments 10 inhibit the binding of an Ot4B7 integrin to one or more of its ligands (e.g., the mucosal MAdCAM addressin (e.g., MAdCAM-1), fibronectin). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a unique antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to readily crystallize.
Pepsin treatment generates an F(ab')2 fraction that has two binding sites and is still capable of cross-linking with an antigen. "Fv" is an antibody fragment consisting of a dimer of a heavy chain variable domain and a light chain variable domain in non-covalent association. The Fab fragment still contains the constant domain of the light chain and the first conslanle domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy-chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation here for Fab' and that the cysteine residues of the constant domains have at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are still known. "Single-chain Fv" or "SCFv" antibody fragments comprise the Vh and V1 domains of antibody, wherein these domains are present in a single polypeptide chain. In one aspect, the Fv polypeptide further comprises a polypeptide linker between the Vfj and V1 domains which allows the scFv to form the desired structure for antigen binding. For a review of SCFV see Pluckthun in The Pharmaco/ogy of Monoc/ona/Antibodies, vol. 11 3, Rosenburg and Moore eds., Springer-Verlag, New York, pp-
269-315(1994) The term "diabodies" refers to small antibody fragments with two antigen-binding silices, which fragments comprise a variable chain domain CYrú connected to a variable light domain (Vl.) on the same polypeptide chain (Vh-Vl).
5 Using a linker that is too small to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93/11611; and Hollinger et al., Proc. Natl.
Academic Sci. USA, 90:6444-6448 (1993). 10 A "complete antibody" is one which comprises a berri antigen-binding variable region such as a light chain constant domain (Cl) and heavy chain constant domains, Chl, C6 and Ch3. The constant domains can be native sequence constant domains (for example, human native sequence constant domains) or amino acid sequence variants thereof. In one aspect, the complete antibody 15 has one or more effector functions. A "variant amino acid sequence" antibody herein is an antibody with an amino acid sequence that differs from the main species antibody. Ordinarily, amino acid sequence variants will have at least about 7°/o, at least about 80°/o, at least about 85°/o, at least about 90°/o, or at least about 90°/o. /q of homology with the main species antibody. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions within or adjacent to the main species antigenic amino acid sequence, but retain antigenic activity. of the antibody constant Tegios will have less effect on antigen binding activity than variations in the variable regions. In the variable regions, amino acid sequence variants will be at least about 90% homologous, at least about 9S'/j homologous, at least about 97% homologous, at least about 98% homologous, or at least about 97% homologous, or at least about 97% homologous. 99% homologous with the main species antibody. "Homology" is defined as the percentage of residues in the 30 amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent homology. Compiler methods and programs for alignment are well known in the art. A "therapeutic monoclonal antibody" is an antibody used for therapy of a human subject.
Therapeutic monoclonal antibodies disclosed herein include anti-a4B7 antibodies. A "glycosylation variant" antibody here is an antibody with one or more carbohydrate fractions attached to it that differ from one or more carbohydrate fractions.
5 linked to an antibody of the main species.
Examples of glycosylation variants herein include antibody with a G1 or G2 oligosaccharide structure, rather than a GO oligosaccharide structure, linked to an Fc region thereof, antibody containing one or two carbohydrate moieties linked to one or two chains thereof. , antibody without carbohydrate linked to one or two antibody heavy chains, etc., and combinations of 10 glycosylation alterations. Antibody "Effector Functions" refer to those antibody activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; binding to Fc receptor;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg, B cell receptor; BCR), and the like.
Depending on the amino acid sequence of the constant domain of their heavy chains, complete antibodies can be designated as different "classes". There are five major classes of complete antibodies: JgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 . The heavy chain constant domains that correspond to the different classes of antibodies: are called cl, Ò, 8, 7, and µ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The "light chains" of antibodies from any vertebrate species can be designated as one of two clearly distinct types, chamfered kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages ) recognize bound antibody to a target cell and subsequently cause lysis of the target cell.
The primary cells to mediate ADCC, NK cells, express only
FCj'RIIj, whereas monocytes express FcRI, FcyRI] and FcRIIL Expression of FCR in hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu.
Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in US patents 5,500,362 or 5,821,337 can be performed.
Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al.
PNAS (USA) 95:652-656 (1998). 10 The terms "Fc receptor" or "FCR" are used to describe a receptor that binds to the Fc region of an antibody.
In one aspect, the FCR is a native human sequence FCR.
In another aspect, the FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses FcRI, FcRII, and FqRjIj, including allelic variants and alternatively spliced forms of these receptors.
FcRII receptors include FcRIJA (an "activation receptor") and FcRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains.
Activation receptor FcyRIIA contains an activation motif based on an immunoreceptor tyrosine (ITAM) in its cytoplasmic domain.
Receptor inhibition FcRIIB contains an inhibition motif based on an immunoreceptor tyrosine (ITIM) in its cytoplasmic domain. (See, review in M.
Daeron, Annu.
Rev.
Immunol. /5:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Ánnu.
Rey. Immunol 9:457-92 (1991); Capel et al., /mmunome/hods 4:25-34 (1994); and from Haas et al. Lab.
Clin.
Med. 26:33-41 (1995). Other FcRs, including those: identified in the future, are included by the term "FcR" here.
The term also includes the neonatal receptor, FcRn, which is responsible for transferring the maternal IgGs to the fetus (Guyer et al., ./. Immunol. ll 7:587 (1976) and Kim et al., J. /mmuno/. 24:249 (1994)). The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
The general hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the chain variable domain light and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences o/'Proteins q//mnnmological /n/eres/, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those
" residues of a "hypervariable dc" (eg residues 26-32 (L 1), 50-52 (L2) and 91-96 (1-3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain, Chothia and Lesk J Mòl.
Biol. 96:901-917 (1987)) - "Framework region" or "FR" residues are those whose variable domain residues in addition to the 5 hypervariable region residues are defined herein.
The hypervariable region or the CDRs thereof can be transferred from one antibody chain to another or to the other protein to confer antigen binding specificity to the resulting antibody (composite) or binding protein - "Humanized" forms of non-human antibodies (by example, rodents) are 10 chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunolobulins (receptor antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region from a non-human species (donor antibody) such as mouse, rat , rabbit or non-human primate having the desired specificity, affinity and capacity.
In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody.
These modifications are made to further refine antibody performance.
In general, the humanized antibody 20 will comprise substantially all of at least one, and typically two variable domains, wherein all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRS are those of a human immunoglobulin sequence. The humanized antibody optionally will further comprise at mencis a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., iVa/ure 332:323-329 (1988); ¢ Provides, Curr.
Op.
No. Biol. 2:593-596 (1992). A "mature affinity" antibody is one with one or more changes in one or more hypervariable regions thereof that result in an improvement in the antibody's affinity for the antigen, compared to a parent antibody that does not have those changes.
In one aspect, mature affinity antibodies will have nanomotar or even picomolar affinities for the target antigen.
Affinity mature antibodies are produced by procedures known in the art.
Marks et al.
Bio/Techno/ogy 10:779-783 (1992)
19/91 describes atinity maturation by VH and VL domain shuffling. Random mutation of CDR and/or framework residues is described by: Barbas et al. Proc Ncu. Ac'ad. Sci, US 91:3809 -381 3 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.
155:1994-2004 (1995); Jackson et al., L Immunol. 154(7):3310-9 (1995); and Hawkins et al., 5 J. Mol. Biol. 226:889-896 (1992).
An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of protein as determined by the Lowry method, and alternatively, greater than 99°4 by weight, (2) to a degree sufficient to obtain at all. minus 10 15 N-terminal residues or internal amino acid sequence by the LlSO of a spin cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within the recombinant cell since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step- "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those who are already ill as well as those in whom the disease or its recurrence must be prevented. Thus, the patient to be treated here may have been diagnosed as having the disease or may be predisposed or susceptible to the disease. The terms "patient" and "subject" are used interchangeably herein. The antibody that is formulated is substantially pure and desirably substantially homogeneous (i.e., free of contaminating proteins, etc.). "Substantially pure" antibody means a composition comprising at least about 25-90-4 antibody by weight, based on the total weight of protein in the composition, at least about 9S'/q or 97% by weight. "Substantially homogeneous" antibody means a composition comprising protein wherein at least about 99% by weight of the protein is specific antibody, e.g., anti-o4B7 antibody, based on the total weight of the protein.
"Clinical remission" as used herein with reference to subjects with ulcerative colitis 30 refers to a full Mayo score of 2 or less points and no individual subscore greater than 1 point. "Clinical remission" of Crohn's disease refers to a CDAI score of 150 points or less.
A "clinical response" as used herein with reference to subjects with ulcerative colitis refers to a reduction in full Mayo score of 3 or more points and 3°/, from baseline, (or a partial Mayo score of 2 or more points and 25°4 or more from baseline if full Mayo score was not performed at visit) with an accompanying reduction in rectal bleeding subscore of 1 or more points or absolute rectal bleeding score 5 of 1 or fewer points. A "clinical response" as used herein with respect to subjects with Crohn's disease refers to a reduction of 70 points or more in the CDAJ score from baseline (Scmana 0).
"Mucosal healing" as used herein in connection with subjects with ulcerative colitis refers to an endoscopic subscore of 1 point or less.
As used herein, "treatment failure" refers to a worsening of the disease, a need for rescue medications, or surgical intervention to treat ulcerative colitis or Crohn's disease. A rescue medication is any new medication or any increase in the dose of a baseline medication required to treat new or unresolved symptoms of ulcerative colitis or Crohn's disease (in addition to antidiarrheals or chronic diarrhea).
Formulations As described herein, it has been found that anti-a4B7 antibodies are highly stable when in a dry formulation, for example, lyophilized with an excess (on a molar basis) of non-reducing sugar. In particular, lyophilized formulations in which the ratio of non-reducing sugar to anti-ct4B7 antibody (mol:mol) is greater than 600:1 are shown here to be stable for at least 2 years.
The present invention provides, in a first aspect, a stable antiLL4B7 antibody formulation. In one aspect, the formulation comprises a buffer, at least one stabilizer and anti-«4B7 antibody. In one aspect, a dry formulation comprises one or more non-reducing sugars and an anti-a4(37 antibody, wherein the ratio of non-reducing sugar to anti-a4P7 antibody (mol:mol) is greater than 600:1. The formulation further comprises one or more free amino acids. One or more amino acids can act as a buffer. In one aspect, one or more of the amino acids can act as a stabilizer.
The formulation may optionally further comprise at least one surfactant. In one embodiment, the formulation is dried, for example, lyophilized. The antibody in the formulation may be a complete antibody or an antigen binding fragment thereof, such as a Fab, Fv, SCFV, Fab' or F(ab')2 fragment. The formulation may contain any desired non-reducing sugars. In one aspect, non-reducing sugars that can be included in the formulation include, for example, mannitol, sorbitol, sucrose, trehalose, raffinose, stachyose, tenelezitose, dexirane, maltitol, lactitol, isornaltubse, palatinit and combinations thereof.
In another aspect, non-reducing sugars are sucrose, trehalose, mannitol, and sorbitol.
The absolute amount of
5 non-reducing sugar in the formulation is not critical, but the ratio of non-reducing sugar to anti-ü4j37 antibody (mol:mol) is greater than 400:1 In another aspect, the ratio of non-reducing sugar to anti-a4B7 antibody ( mol:mol) is at least about 600:1; at least about 625:1; at least about 650:1; at least about 675:1, at least about 700:1; at least about 750:1, at least about 800:1, at least about 1000:1, at least about 1200:1, at least about 1400:1, at least about 1500:1, at least about 1600:1, at least about 1700:1, at least about 1800:1, at least about 1900:1, or at least about 2000:1. Generally, it is desirable that the non-reducing sugar is present in an amount that reduces soluble aggregate formation in a liquid formulation, as aggregate formation occurs.
Í5 freezing and thawing and/or drying and reconstitution.
A ratio of non-reducing sugar to anti-u437 antibody (mol:mol) greater than about 730:1 can generate a slightly reduced soluble aggregate formation in the lyophilized state.
The sugar:protein ratio can be greater than 1.5:1 (w/w). In another aspect, the non-reducing sugar concentration for liquid formulations (e.g., pre-drying or post-reconstitution) is in the range of from about 10 mM to about 1 M, e.g., from about 60 mM to about 600 mM, about 100 mM to about 450 mM, about 200 mM to about 350 mM, about 250 mM to about 325 mM, and about 275 mM to about 300 mM.
In another aspect, the amounts of non-reducing sugar in a dry (e.g., lyophilized) formulation are in the range of from about 40°C to about 70°4 (w/w of dry formulation). In another aspect, the amounts of non-reducing sugar in a dry (e.g., lyophilized) formulation are in the range of from about 40% to about 60°4, from about 45°4 to about 55°4 or about 51% (w/w). In other aspects, the amounts of non-reducing sugar in a dry formulation (eg, lyophilized) is greater than about 5 1'/0 (w/w of dry formulation) when the protein amount is about 31% ( dry formulation w/w)
or greater than about a 1.6:1 mass ratio of non-reducing sugar to protein in the dry formulation.
In yet another aspect, sucrose is the non-reducing sugar for use in the formulation.
The formulation may contain any desired or free amino acid, which may be in L-form, D-form or any desired mixture of these forms. In one aspect, free amino acids that can be included in the formulation include, for example, histidine, alanine, arginine, glycine, glutamic acid, serine, lysine, tryptothane, valine, cysteine and combinations thereof. Some amino acids can stabilize proteins against degradation during manufacturing, drying, lyophilization and/or storage, for example, through hydrogen bonds, salt bonds, antioxidant properties, or hydrophobic interactions or by exclusion from the protein surface. Amino acids can act as tonicity modifiers or can act to reduce the viscosity of the formulation. In another aspect, free amino acids, such as histidine and arginine, can act as cryoprotectants and lyoprotectants, and do not crystallize when lyophilized as components of the formulation. Free amino acids such as glutamic acid and histidine, alone or in combination, can act as buffering agents in aqueous solution in the pH range of 5 to 7.5. In yet another aspect, free amino acid concentrations for liquid formulations are in the range of about LO mM to about 0.5 M, for example, from 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. . In yet another aspect the amounts of histidine in a dry formulation (e.g., lyophilized) are in the range of from about 1°/o to about 10°/o (w/w of dry formulation), or from about 3° /) to about 6°/, (w/w). In some embodiments, the amount of histidine in a dry formulation (eg, lyophilized) is greater than about 4°4 (w/w of the dry formulation) when the amount of protein is about 31% (w/w of the dry formulation) or greater than about a 0.15:1 mass ratio of histidine to protein in the dry formulation. In yet another aspect, the amounts of aTginin in a dry formulation (e.g., lyophilized) are in the range of from about 4°/q to about 20°4 (w/w of dry formulation), or from about 10% to about 1S°/, (w/w). In some embodiments, the amount of arginine in a dry formulation (eg, lyophilized) is greater than about 13% (w/w of the dry formulation) when the amount of protein is about 3 1°/, (w/ p of the dry formulation) or greater than about a 0.4:1 mass ratio of arginine to protein in the dry formulation. In the combination embodiments of amino acids such as histidine and arginine, the molar ratio of total amino acid to antibody can be at least 200:1, about 200:1 to about 500:1, or at least 400:1.
The formulation may optionally further contain at least one surfactant. In one aspect, surfactarites that can be included in the formulation include, for example,
" polysorbate 20, polysorbate 80, a poloxamer (Pluronic®) and combinations thereof.
When present, the surfactant is generally included in an amount that reduces the formation of insoluble antibody aggregates, for example, during bottling, freezing, drying, lyophilizing and/or reconstituting. The concentration of the surfactant, for example, in a pre-dried, (e.g., lyophilized) or post-reconstitution formulation, is generally from about 0.0001% to about 1.0%, from about 0.01 °4 to about 0.1%, for example about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08.% or 0.09% (jp/v), 0.05% at 0.07°4 or 0.06% (jp/v). The amount of surfactant, eg in a dry formulation (eg lyophilised), is generally from about 0.01°4 to about 3.0%, (w/w), from about 0.10% at about 1.0°/o, for example about 0.15%, 0.20°4, 0.25%, 0.30°/1, 0.35°/), 0.40% , or 0.50% (w/w). In another aspect, the surfactant:antibody molar ratio is about 1:1. The anti-c4f17 antibody can be present in any desired amount in the formulation, as long as the ratio of non-reducing sugar to anti-ot4[37 antibody (mol:mol) is greater than about 600:1. However, the formulation may contain a high concentration of anti-a4[IL7] antibody. For example, liquid formulations can comprise at least about 10 mg/ml, at least about 20 mg/ml, at least about 30 mg/ml, at least about 40 mg/ml, at least about 50 mg µg/ml, at least about 60 nil/ml, at least about 70 mg/ml, at least about 80 mg/ml, at least about 90 mg/ml, at least about 100 mg/ml, or about from 40 mg/ml to about 80 mg/ml of anti-cL4B7 antibody, about 60 mg/ml of anti-a4B7 antibody. Dry formulations (e.g., lyophilized) may contain at least about 5°/q, at least about 10°4, at least about 15°4, at least about 20°4, at least about 2°/ , at least about 30%, or about 3 µ/o or about 32 µ/, anti-α4 antibody by weight. If desired, the formulation may further comprise a metal chelator and/or an antioxidant, as other pharmaceutically acceptable excipients. Suitable metal chelators include, for example, methylamine, ethylenediamine, despheroxamine, trientine, histidine, malate, phosphonate compounds, for example, etidronic acid, ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and the like. Suitable antioxidants include, for example, citric acid, uric acid, ascorbic acid, lipoic acid, glutathione, rocorol, carotene, lycopene, cysteine and the like. The formulation can be a liquid or a solid. Liquid formulations can be aqueous solutions or suspensions, prepared in an appropriate aqueous solvent such as water.
or an aqueous/organic mixture, such as mixtures of alcohol and water. Liquid formulations can have a pH of from about 5.5 to about 7.5, from about 6.0 to about 7.0, or from about 7.0. of 6.0 and about 6.5, such as about 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5 Liquid formulations may be refrigerated (eg 2 -8°C), or frozen (eg at -20°C 5 or 80°C) for storage Solid formulations may be prepared in any suitable manner and may be in the form of a pie or powder, for example. Solid is prepared by drying a liquid formulation as described herein, eg by freeze drying, spray drying, air drying to a film (eg for transdermal delivery), mixing into a lipid emulsion and drying as 10 to spheres for oral release or film for transdermal release. When the formulation is a solid formulation, the formulation may have a moisture content of no more than about 5°/, no more than about 4.5% no more than about 4°4, no more than about 3.5°4, no more than about 3°/0, no more than about 2.5%, no more than about 2%, no more than about 1.5%, no more than about 1%, or is substantially anhydrous. Solid formulations can be dissolved, that is, reconstituted, in an appropriate solvent OL1 medium to become a suitable liquid for administration. Suitable solvents for reconstituting the solid formulation include water, isoline, buffer, e.g., phosphate buffered saline, Ringer's solution (lactate or dextrose), minimal essential medium, alcohol/aqueous solutions, dextrose solution, etc. The amount of solvent can result in a therapeutic protein concentration greater than, equal to or less than the concentration prior to drying. In one aspect, the concentration of reconstituted anti-u437 antibody is the same concentration as in the pre-drying liquid formulation.
The formulation may be sterile, and it may be obtained in accordance with procedures known to those skilled in the art to generate pharmaceutical formulations suitable for administration to human subjects, before or after preparation of the formulation. The formulation can be sterilized as a liquid, for example, before drying and/or after reconstitution by small pore filtration, by aseptic processing or by exposure to ultraviolet radiation. Filter pore sizes can be 0.1 µm or 0.2 µm to filter micro-organisms or 10 to 20 nm to filter viral particles. Alternatively, or in addition, the dry formulation can be sterilized, for example, by exposure to gamma radiation. In one aspect, the liquid anti-a4[37] antibody formulation is filter sterilized prior to drying.
In one aspect, the formulation is storage stable. In another aspect, the formulation is stable on storage in the dry state.
Stability can be tested by evaluating the physical stability, chemical stability, and/or biological activity of the antibody in the formulation around the time of formulation as well as after storage of noted temperatures.
Physical and/or chemical stability of a liquid or powder formulation
The reconstituted dried can be evaluated qualitatively and/or quantitatively in a variety of different ways (see, for example, Ana/ytica/ Techniques for BiopharmyaceM/ical Deyelopment, Rodriguez-Oiaz et al. eds.
Jnforma Healthcare (2005)), including assessment of aggregate formation (eg using size exclusion chromatography (or gel filtration) (SEC), time-of-flight mass spectrometry in matrix-assisted laser desorption ionization ( MALDI-J'OF MS), analytical ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MAI.LS)), microscopic imaging based on flux, electronic impedance (coulter), light obscuration or other liquid particle counting system, when measuring 15 turbidity, by density gradient centrifugation and/or by visual inspection); evaluating charge heterogeneity using cation exchange chromatography (see further VIasak and Ionescu, Curr.
Pharm. .Biotechnol. 9:468-481 (2008) and Harris et al. J.
Chromatography B {
Biomed.
Sci.
Appl. 752:23 3-245 (2001)), isoelectric focusing (IEF), for example capillary technique (cIEF), or capillary zone electrophoresis, amino-terminal or carboxy-terminal sequence analysis; inassa spectrometry analysis, SDS-PAGE or SEC analysis to compare fPaµnted, intact, and multimeric (ie, dimeric, trimeric, etc.) antibody; peptide niapa (for example, tryptic or LYS and the like); evaluated biological activity or antigen-binding function of the antibody; and the like.
Biological activity or antigen-binding function, e.g., binding of the anti-cL4B7 antibody to MAdCAM (e.g., MMCAM-L) or inhibition of binding of a cell expressing α7 would integrate to MAdCAM (e.g. , MAdCAM-1), for example, immobilized MAdCAM (for example, MAdCAM-1), can be evaluated using various techniques available to the skilled practitioner (see, for example, Soler et al., ./ Pharmacol.
Expert
The R. 330:864-875 (2009)). The stability of a solid state formulation can further be assessed qualitatively and/or quantitatively in a variety of ways, including direct tests such as crystal structure identification by X-Ray Powder Diffraction (XRPD); evaluation of the structure of the solid state antibody using spectroscopy of
Fourier Transform Inflared (FTIR); and measurement of thermal transitions in the freeze-dried solid (melting, glass transition, etc.) using Differential Scanning Calorimetry (DSC, for example, to assess denaturation) and indirect tests such as moisture content measurement by Karl Fisher test, for example , to extrapolate the possibility of chemical instability through hydrolysis.
Measuring the moisture content of a dry formulation can indicate how likely a formulation will undergo chemical or physical degradation, with higher moisture leading to further degradation.
Stability can be measured at a selected temperature for a selected period of time.
In one aspect, a dry (e.g., lyophilized) formulation is stable at about 40°C, 78°/j RH for at least about 2-4 weeks, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, or at least about 18 months.
In another aspect, the formulation (liquid or dry (eg, lyophilized)) is stable at about 5'C and/or 25'C and 60% RH for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, or at least about 48 months.
In another aspect, the formulation (liquid or dry (eg, lyophilized)) is stable at about -20°C for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about ] 8 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.
In addition, the liquid formulation may, in some embodiments, be stable after freezing (a, for example, RO'C) and melting, as, for example, after 1, 2 or 3 freeze-thaw cycles.
Instability may involve any one or more of: aggregation (eg, non-covalent soluble aggregation (caused by hydrophobic or charge interactions), covalent soluble aggregation (eg, disulfide bond rearrangement/scattering), insoluble aggregation (caused by denaturation of protein at the liquid/air and liquid/solid interfaces), deamidation (eg Asn deamidation), oxidation (eg Met oxidation), isomerization (eg Asp isomerization), denaturation, shear/hydrolysis/fragmentation (eg fragmentation in hinge region), succinimide formation, N-terminal extension, C-terminal processing, glycosylation differences, and semethants.
A stable formulation can contribute to the low immunogenicity of an anti-Q4B7 antibody. An immunogenic anti-a4B7 antibody can lead to a human-anti-human antibody (HAHA) response in human subjects or patients. Patients who develop a HAHA response to an anti-c4j37 antibody may have adverse events (eg, site of infusion reaction) on treatment or may eliminate anti-c4j37 antibody rapidly, resulting in a lower dose than planned by the treatment. A report (Feagen et al. (2005) N. Engl. J. Med. 352:2499-2507) of an early study of an anti-u4B7 antibody treatment indicated that anti-human antibodies developed by scman 8 in 44% of patients treated patients. The antibody in this study was 10 stored as a liquid and contained no polysorbate.
In some embodiments, the formulation can increase the proportion of HAHA-negative patients by at least 40%, at least 50°/q, at least 60%, at least 70°4, at least BO'/o, or at least 90% of the patients in comparison with the HAHA results of a less stable formulation.
In some embodiments, an anti-u4B7 antibody formulation has Z 50°4 of major isoform charged, Z 55°4 of major isoform charged, or 65 to 70°4 of major isoform charged. In other aspects, a stable anti-α4[37] antibody formulation has <45% acid charged isoforms, <40% acid charged isoforms, <30%/q acid charged isoforms, or 22 to 28% acidic isoforms. In still other aspects, a stable anti-q4B7 antibody formulation has <25°4 basic isoforms, <20°4 basic isoforms, <15% basic isoforms, about 5/0 basic isoforms, or about 10 % of basic isoforms. In one aspect, a stable anti-Z4B7 antibody formulation has Z 55'4 of major isoforms, S 30'4 of acidic isotomes and/or < 20% of basic isoforms, for example, as determined by CEX. In another aspect, a stable anti-cL4B7 antibody formulation has Z50%/q of major isoform, <45% of acidic isoforms, and/or <10% of basic isoforms, for example, as determined by CIEF.
In some aspects, a solid, dry formulation of anti-u4B7 antibody has £10°/q moisture content, <S°/, moisture content, or <2.5% moisture content. The time required for reconstitution is Z60 minutes, <50 minutes or ,<40 minutes or <30 minutes or <20 minutes.
Monomeric content and/or aggregate content (for example, as dimers, trimers, tetramers, pentamers, oligomers and higher order aggregates), that is, in the liquid formulation, or in a dry formulation after reconstitution, it can be measured by SEC,
"MALDI-TOF MS, analytical ultracentrifugation, light scattering (DLS or MALLS), or nanoscale measurement such as NTA nanoparticle tracking analysis, NanoSight Ltd, Wiltshire, UK) Resolution, characterization and quantification of aggregate can be achieved in aggregate resolution, characterization and quantification can be achieved in aggregate. a number of ways, including increasing the length of separation by 5 SEC column, for example, by a larger column or by serially linking one or more SEC columns in line with the initial analytic SEC column, complementary SEC quantification of monomers with light scattering, or using NTA.
In one embodiment, an anti-α4j37 antibody formulation is Z 90% monomeric antibody, Z95% monomeric antibody, or 97 to 99% monomeric antibody 10 . In another embodiment, most of the material in an anti-cL4B7 antibody formulation has an average radius of 20 nm, 15 nm, 10 nm, or about 5 to about 7 nm. In one aspect, an anti-«4f17 antibody formulation has Z 80% of the amount of light heavy chain by protein analysis. In one aspect, there is >90°/q of lighter heavy chain. In another aspect, an anti-a4[37] antibody formulation has <10°4 aggregate, £5/) aggregate, ,< 2.5% aggregate, < 1.S°/j aggregate, < 1 ,O'/o of aggregate or £0.5% of aggregate. In another aspect, the stable anti-cL4B7 antibody formulation has >96°4 monomer and/or <2.5% aggregate. In yet another aspect, a stable anti-a4B7 antibody formulation has about 99°4 monomer and/or about <1°/aggregate.
Particle sizes, for example, of aggregates or undissolved excipient, i.e., in the reconstituted formulation, can be measured by obfuscation (eg, Liquid Particle Counting System (HIAC) pqt Hach Ultra Analytics (Grants Pasx, OR)), microscopy, coulter counter, or digital (eg, flow-based) microscopic imaging-based system such as microfluidic imaging (MFI) by Brightwell (Ottawa, CA) or FLOWCAM® imaging particle analyzer by Fluid Imaging Technologies (Yarmoulh, ME) . In one aspect, particle size in an anti-u4j37 antibody preparation is about 30 µm, about 25 µm, about 10 µm, about 5 µm, about 2 µm, or 1 µm or less. The amount of particles must be minimized in antibody formulations. In one aspect, the anti-cy4f7 antibody formulation has less than < 6000 particles > 10 µm and/or less than 600 particles Z 25 µm in one dose (U.S.
Pharmacopoeia Chp. 788, obscuration counting method; half of those amounts by microscopic quantification method). In yet another aspect, an amount of particles per milliliter, e.g., by MFI measurement, in a dose of an anti-a4B7 antibody formulation, e.g., reconstituted formulation is about 500 to about 2000 or about 1000 to about about 3000 of 2-10 µm particles per ml, about 50 to about 350 z10 µm particles per ml, and about 0 to about 50 25 µm Z particles per ml.
In one embodiment, an anti-mx4j37 antibody formulation has a binding affinity of about 60% to about 140% of the anti-a4B7 reference standard antibody. In one aspect, an anti-a4B7 antibody in a formulation described herein binds to a4j37, for example, in a cell (WO98/06248 or US patent 7,147,851), at a value of about 80% to about 120% of the reference standard. In another embodiment, an anti-α4j37 antibody formulation is capable of inhibiting at least 50%, or at least 60% of the binding of a cell expressing α4β17 integrin to MAdCAM, e.g., MAdCAM-1, to MAdCAM-Ig quiniera (See US patent application publication 20070122404, further for reference standard examples). As noted above, freezing the formulation is specifically contemplated here. 'Thus, the formulation can be tested for stability in freezing and melting. Thus, the antibody in a liquid formulation may be stable upon freezing and thawing of the formulation, for example, the antibody may be stable after one, two, three, four, five or more freeze/thaw cycles. In some embodiments, formulation C is a liquid formulation comprising at least about 50 mg/ml to about 100 mg/ml of anti-C4[37 antibody, a buffering agent (e.g., histidine), and at least about 9 °/1 (w/w) of non-reducing sugar (eg sucrose, trehalose or mannitol). In one embodiment, the formulation comprises at least about 50 to about 80 mg/ml, about 60 mg/ml of anti-a4P7 antibody, a buffering agent (eg, histidine), a free amino acid (eg, arginine ) and at least about 9°/) or 10°4 (w/w) non-reducing sugar (eg, sucrose, trehalose or mannitol). In another embodiment, the formulation comprises at least about 60 mg/ml of anti-α437 antibody, a buffering agent (eg, histidine), a free amino acid (eg, arginine) and at least about 10°4 ( w/w) of non-reducing sugar (eg sucrose, trehalose or mannitol). In said embodiments, the buffer concentration is about 15 to about 75 mM, about 25 to about 65 mM, or about 50 mM. The concentration of free amino acid is about 50 to about 250 mM, about 75 to about 200 mM, about 100 to about 150 mM, or about 125 mM.
In one embodiment, the formulation is a solid scca formulation (eg, a lyophilized formulation), comprising a mixture of a non-reducing sugar, an anti-a4[37 antibody, histidine, arginine, and polysorbal 80, and the molar ratio of non-reducing sugar for anti-a4|37 antibody (mol:mol) is greater than 600:1.
In another embodiment, the formulation is a dry, solid, amorphous formulation (eg, a lyophilized formulation), comprising a mixture of non-reducing sugar, an anti-α4j37 antibody, histidine, arginine, and polysorbate 80, and the molar ratio of non-reducing sugar for anti-«4j37 antibody (mol:mol) is greater than 600:1.
In one embodiment, the formulation is a lyophilized formulation comprising a non-reducing sugar, an anti-á4j37 antibody, histidine, arginine, and polysorbate 80, and the molar ratio of non-reducing sugar to anti-cL4B7 antibody (mol:mol) in the formulation is greater than 600:1.
In one embodiment, the formulation is a lyophilized formulation comprising a non-reducing sugar, an anti-«4|37 antibody, histidine, arginine, and polysorbate 80, wherein the molar ratio of non-reducing sugar to anti-a4fl7 aritibody (mol:mol) ) in the formulation is greater than 600:1 and the molar ratio of arginine to anti-a4B7 antibody (mol:mol) in the formulation is greater than 250:1.
In one term, the formulation is a liquid formulation and comprises at least about 60 mg/ml anti-c7 antibody, at least about 10°/q (w/v) non-reducing sugar, and at least about 125 mM of one or more free amino acids.
In one modality, the formulation is a liquid formulation and comprises at least about 60 mg/ml anti-u4[37] antibody, b. about 175 mM of one or more free amino acids.
In one embodiment, formulation C is a liquid formulation and comprises from about 60 mg/ml to about 80 mg/ml anti-α437 antibody, a swelling agent and at least about 10-4 (w/w) sugar.
In one embodiment, the formulation is a liquid formulation and comprises from about 60 mg/ml to about 80 mg/ml anti-a4B7 antibody, histidine and at least about 10% (w/w) sucrose.
In one modality, the formulation is lyophilized and prepared as a single dose in a fiasco. The vial is desirably stored at about 2-8°C until it is administered to a subject in need thereof. The bottle can, for example, be a 20 or 50 cm' bottle (for example for a dose of 60 mg/ml). The vial can contain at least about 120 mg, at least about 180 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 540 mg, or at least about 540 mg. 900 mg of anti-«4B7 antibody. In one aspect, the vial contains about 300 mg of anti-0t4B7 antibody.
5 One or more pharmaceutically acceptable carriers, excipients or stabilizers as described in Reming/on. The Science and Practice of Pharmacy, 21st Edition, Hendrickson, R. Ed. (2005) may be included in the formulation provided as long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include; additional buffering agents; co-solvents; antioxidants including ascorbic acid and melionin; chelating agents such as EDTA; metal complexes (eg, Zn protein complexes); biodegradable polymers such as polyethers; condoms; and/or salt-forming counterions such as sodium. a4P7 Antibodies Suitable anti-«²4f37 antibodies for use in the formulations include antibodies from any desired source, such as fully human antibodies, nurine antibodies, rabbit antibodies, and the like, and any desired modified antibodies, such as chimeric antibodies, humanized antibodies, and the like. Amino-binding fragments of any of these types of antibodies, such as Fab, Fv, SCFV, Fab' and F(ab')2 fragments, are further suitable for use in the formulations. The anti-α4j37 antibody can bind to an epitope on the α4 chain (eg, humanized MAb 21.6 (Beridig et al., US Patent 5,840,299)), on the β37 chain (eg, FJB504 or a humanized derivative (eg Forig et al, paterite US 7,52&236)), naked to a combinatorial epitope formed by the association of a 4 strand with the p7 strand. In one aspect, the antibody binds to a combinatorial epitope on the a4B7 complex, but does not bind to an epitope on the cy4 chain or the B7 chain unless the chains are in association with each other. The association of α4 integrin with β37 integrin can react to a combinatorial epitope, for example, by bridging the proximity residues present in both strands that together comprise the epitope, or by exposing conformationally to a strand, for example, to 4 integrin chain or 37 integrin chain, an epitope binding site that is inaccessible for antibody binding in the absence of the appropriate integrin partner or in the absence of integrin activation. In another aspect, the anti-α4j37 antibody binds to both the α4 integrin chain and the B7 integrin chain, and thus is
"specific for α4j37 integrin complex. Such antibodies may bind α4β37 but do not bind α4f11, and/or do not bind αEB7, for example. In another aspect, the anti-α4β37 antibody binds to the same or substantially the same epitope as the Act-1 antibody (Lazarovits, AI et al., J. /mmuno/., /33(4): 1857-1862 (1984), Schweighoffer et al., J.
5 /mmuno/., 151(2): 717-729, ]993; Bednarczyk et al., J. Biol. Chem., 269(11):8348-8354, 1994). The ACT-I hybridoma cell line, which produces the murine monoclonal antibody Act-1, was deposited under the provisions of the Budapest Treaty on August 22, 2001, in the name of Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, Mass.
02139, USA, at American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 201 10-2209, U.S.A., under Accession No. PTA-3663. In another aspect, the anti-a4f17 antibody is a human antibody or a 4|37-binding protein using the CDRs provided in US patent application publication 20i 0/0254975.
In one aspect, the anti-α4j37 antibody inhibits the binding of ct4[37 to one or more of its ligands (e.g., the mucosal addressin, e.g., MAdCAM (e.g., MAdCAM-1), fibronectin, and/or vascular addressin (VCAM)). Primate MACJCAMs are described in PCT publication WO 96/24673, the entire teaching of which is incorporated herein by this reference. In another aspect, the anti-α4[37] antibody inhibits the binding of C17 to MAdCAM (e.g., MAdCAM-I) and/or fibronectin without inhibiting VCAM binding.
In one aspect, the anti-u4[37 antibodies for use in the formulations are humanized versions of the mouse Act-1 antibody. Appropriate methods for preparing humanized antibodies are well known in the art. Generally, the humanized antibody will contain a heavy chain which contains the 3 determining regions of heavy chain complementarity (CDRS, CDRI, SEQ ID NO:8, CDR2, SEQ ID NO: 9 and CDR3, SEQ JD NO:10) of the mouse Act-1 antibody and appropriate human heavy chain framework regions; and further contains a light chain which contains the 3 light chain CDRs (CDRI, SEQ ID NO:11, CDR2, SEQ JD NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1 antibody and chain framework regions take appropriate human. The humanized Act-1 antibody may contain any appropriate human framework region, including consensus framework region, with or without amino acid substitutions. For example, one or more of the framework amino acids can be replaced with another amino acid, such as the amino acid at the corresponding position in the mouse Act-1 antibody. The human constant region or portion thereof, if present, can be derived from the k or X light chains, and/or the y heavy chains (for example, j'l, y2, y3, y4), µ, a (for example , cil, cc2), b or e of human antibodies, including allelic variants.
A particular constant region (eg LgGl), variable or parts thereof can be selected to configure the effector function.
For example, a mutated constant region 5 (variant) can be incorporated into a fission protein to minimize binding to Fc receptors and/or ability to release complement (see, for example, Winter et al., GB 2,209,757 B; Morrison et al. al., WO 89/07142; Morgan et al., WO 94/29351, Dcc. 22, 1994). Humanized versions of antibody Act-] have been described in PCT publications WO98/06248 and WO07/61679, the entire teachings of each of which are incorporated herein by this reference.
In another aspect, humanized anti-α4|37 antibodies for use in the formulation comprise a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO:2, and a light chain variable region comprising amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132 of SEQ ID NO:5. If desired, an appropriate human constant region may be present.
For example, the humanized anti-«4j37 antibody can comprise a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:2 and a light chain comprising amino acids 21 to 239 of SEQ ID NO:5. In another example, the humanized anti-q4f17 antibody can comprise a heavy chain comprising amino acids 20 to 470 of SEQ ID NO:2 and a light chain comprising amino acids 20 to 238 of SEQ ID NO:4. Figure 4 shows an alignment comparing the generic light chains of human antibodies to murine antibodies.
The alignment illustrates that the humanized light chain of vedolizumab (eg, Chernical Abstract Service (CAS, American Chemical Society) Registry Number 94360966 3), with two mouse residues swapped to human residues, is more human than the chain light weight of LDP-02 (Fif'ura 3). In addition, LDP-02 alway has a hydrophobic, flexible alanine 114 and a hydrophilic (Aspartate 115) site which is replaced in vedolizumab with the threonine 114 residue containing slightly hydrophilic hydroxyl and potentially inwardly facing hydrophobic valine 115 residue .
Other substitutions to the antibody sequence can be, for example, mutations to the heavy and light chain structural regions, such as an isoleucine to valine mutation at residue 2 of SEQ LD NO:14; a methionine to valine mutation at residue 4 of SEQ ID NO:14; an alanine to glycine mutation at residue 24 of SEQ ID NO:15; an arginine to lysine mutation at residue 38 of SEQ ID NO:lS; an alanine to arginine mutation at residue 40 of SEQ TD NO:1S; a methionine to isoleucine inmutation at residue 48 of SEQ ID NO:1S; an isoleucine to leucine mutation at residue 69 of SEQ ID NO:i5; an arginine to valine mutation at residue 7i of SEQ ID NO:lS; a threonine to isoleucine mutation at residue 73 of SEQ ID NO:IS; or any combination of the same; and replacing the heavy chain CDRS with the CDRs (CDRI, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) from the mouse Act-1 antibody; and replacing the light chain CDRS with the light chain CDRs (CDRI, SEQ JD NO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) from the mouse Act-1 antibody.
In some embodiments, humanized anti-a4f17 antibodies for use in the formulation comprise a heavy chain variable region that has about 95%, 96°4, 97°4, 98%, or 99% sequence identity to amino acids 20 to 140 of SEQ LD NO:2, and a light chain variable region having about 9S"/q, 96%, 97%, 98°4, or 99% sequence identity to amino acids 20 to 13 1 of SEQ ID NO: 4 or amino acids 21 to 132 of SEQ ID NO: 5. Amino acid sequence identity can be determined using an appropriate sequence alignment algorithm, such as the Lasergene system (DNASTAR, Ine., Madison, Wis.), using standard parameters .
In one embodiment, the anti-a4B7 antibody for use in the formulation is vedolizumab (CAS, American Chemical Society, Ref. Number 943609-66-3). Still other q4B7 antibodies can be used in the formulations and dosing schedules described herein.
For example, the ü4B7 antibodies described in US 2010/0254975 (Amgen, Inc.), incorporated by reference herein in their entirety, are suitable for use in the formulations and methods of treating inflammatory bowel disease in an individual.
. Anti-q4B7 antibody can be produced by expression of nucleic acid sequences encoding each strand in living cells, e.g., cells in culture.
A variety of host expression vector systems can be used to express the antibody molecules of the invention.
Host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but still represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express an anti-44B7 antibody in situ.
These include, but are not limited to, microorganisms such as bacteria (eg E.co/i, B.subti/is) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing sequences of antibody edification; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, plasmid Ti ) containing antibody coding sequences; or mammalian cell systems (eg, COS, CHO, BHK, 293, 10 3T3, NSO cells) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (per example, the adenovirus late promoter; the vaccinia virus 7.5K promoter). For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells, in conjunction with a vector as the main promoter element of human egallovirus early intermediate gene, is an effective expression system for antibodies (Foeckin{' el al., Gene 45: 101 (1986); Cockett et al., Bio/Technology 8:2 (]990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when a large amount of such a protein must be produced, in order to generate pharmaceutical compositions from an antibody molecule, vectors that direct the expression of high levels of toxin protein products that are readily purified may be desirable. Such vectors induce, among others, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), wherein the antibody coding sequence can be individually linked in the vector in frame with the lac Z coding region 25 such that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nuc/eic Acids Res. 13:3 l6l-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5 509 (1989)); and the like. pGEX vectors can further be used to express foreign polypeptides as fission proteins with glutathione S-transferase (GST). In general, said fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to glutathione-agarose matrix spheres followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or Xa prolease cleavage sites so that the cloned gene product can be released from the GST Faction.
In an insect system, Autographa c'a/ffornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign É'enes.
The virus grows in Spodopcra fmgiperda cells.
The antibody coding sequence can be cloned individually into non-essential regions (eg the polyhedrin gene) of the virus and placed under the control of an ACNPV promoter (eg the polyhedrin promoter). In mammalian host cells, a number of vim-based expression systems can be used.
In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be linked to a transcription/translation control complex, for example, the late promoter and tripartite leader sequence.
This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo.
Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg, see Logan & Shenk, Proc.
At//. Academic
Sci.
USA 81:355-359 (1984)). Specific initiation signals may still be required for efficient translation of inserted antibody coding sequences.
These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading region of the desired coding sequence to ensure translation of the complete insert.
These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
Expression efficiency can be improved by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, Bittner et al., Methods in Enzymol. 153:5 -544 (1987)). Furthermore, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired.
Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function.
Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed foreign protein.
To this end, eukaryotic host cells that possess the cellular machinery for processing the primary transcript, glycosylation and phosphorylation of the gene product can be used.
Such mammalian host cells include, among others, Chinese hamster ovary cells
(CHO), NSO, HeLa, VERY, newborn hamster kidneys (BHK), monkey kidneys (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma cells (eg Hep G2), strains of breast cancer cell such as BT483, Hs578T, HTB2, BT20 and T47D cell line, and normal mammary gland such as CRL7030 and Hs5788st.
The glycosylation machine of different cell types can produce antibodies with different glycosylation composition in addition to another cell type, or without glycosylation, as with bacterial cells.
In one aspect, the cell types for producing the anti-a4B7 antibody are mammalian cells, such as NSO or CHO cells.
In one aspect, mammalian cells can further comprise the deletion of an enzyme involved in cellular metabolism and the exogenous gene of interest can be operably linked to a replacement enzyme, for example, in a construct or vector for introduction into cells, for example , by transformation or transfection.
The construct or vector with the exogenous gene gives cells hosting the construct or vector a selection advantage to encourage production of the polypeptide encoded by the exogenous gene.
In one embodiment, CHO cells are DG44 cells (Chasin and Urlaub (1980) PNAS USA 77:4216), comprising the dektion or inactivation of the dihydrofolate reductase gene.
In another embodiment, CHO cells are CHO K1 cells comprising the deletion or inactivation of the glutamine synthase gene (see, for example, US patents 5,122,464 or 5,827,739). Solid Formulations Solid formulations of the invention are generally prepared by drying a liquid formulation.
Any suitable method of drying can be used, such as lyophilization or spray drying.
Freeze drying involves freezing a liquid formulation, usually in the container that will be used to store, transport and distribute a formulation (eg, a vial). (See, for example, Gatlin and Nail in Protein Puri/ica/ion Process Engineering, ed.
Roger G.
Harrison, Marcel Dekker Inc., 317-367 (1994).) Once the formulation is frozen, the atmospheric pressure is reduced and the temperature is adjusted to allow removal of the frozen solvent, for example, by sublimation.
This step in the freeze-drying process is sometimes referred to as a primary drying.
If desired, the temperature can then be raised to remove any solvent that is still bound to the evaporative dry formulation.
This step in the freeze-drying process is sometimes referred to as secondary drying.
When the formulation has reached the desired degree of drying, the drying process is
is completed and containers are sealed. The final solid formulation is sometimes referred to as a "lyophilized formulation" or a "surface." The freeze-drying process can be carried out using any suitable equipment. Appropriate lyophilization equipment is available from a number of commercial sources (eg, SP Scienlific, 5 Stone Ridge, NY). A variety of suitable apparatus can be used to dry liquid formulations to produce a solid (eg, freeze-dried) formulation. Generally, lyophilized formulations are prepared by those skilled in the art using a sealed chamber that contains shelves, into which the vials of the liquid formulation to be dried are 10 placed. The temperature of the shelves comes as the cooling and heating rate can be controlled, as well as the pressure inside the chamber. It will be understood that various process parameters discussed herein refer to processes performed using this type of apparatus. Those skilled in the art can easily adapt the parameters described here to other types of drying apparatus if desired. 5 Appropriate temperatures and an amount of vacuum for primary and secondary drying can be readily determined by one skilled in the art. In general, Formulation C is frozen at a temperature of about -30°C or less, such as -40°C or -50°C. The rate of cooling can affect the quantity and size of ice crystals in the matrix. Primary is generally conducted at a temperature that is about 10°C, about 20°C, about 30°C, about 40°C or about 50°C warmer than the freezing temperature. , the primary drying conditions can be adjusted to keep the anti-ü4j37 antibody below the glass transition temperature or forrriulation collapse temperature. Above the collapse temperature, the frozen amorphous matrix may flow (collapse), with a result that protein molecules may not be surrounded by a rigid, solid matrix and protein molecules may not be stable in the collapsed matrix. Also, the formulation may be difficult to fully dry if collapse occurs. The resulting larger amounts of moisture in the formulation may lead to higher rates s of protein degradation and a reduction in the amount of time the lyophilized product can be stored before its qualities diminish to unacceptable levels.
In one aspect, shelf temperature and chamber pressure are selected to keep the product temperature below the collapse temperature during primary drying. The vkrea transition temperature of a frozen formulation can be measured by methods known in the art, for example, by differential scanning calorimetry
(DSC). The collapsing temperature can be measured by methods known in the art, for example, freeze-drying microscopy. The ratio of non-reducing sugar to protein (mol:tnol) and the amounts of other formulation components will impact the glass transition temperature and collapse temperature. In some embodiments, a glass transition temperature for an α437 antibody formulation is about -35°C to about -10°C, about -35°C to about -25°C, or about -35 °C to about -29 °C. In another embodiment, the glass transition temperature of a c14B7 antibody formulation is about -29°C. In some embodiments, the glass transition temperature of an a4B7 antibody formulation is about -30°C, about -3°C, about -32°C, about 10 -33°C, about -34 °C, about -35°C or about -36°C. In some embodiments, a collapse temperature of a u437 antibody formulation is about -30°C to about 0°C, about -28°C to about -25°C, or about -20°C to about -10°C. In another embodiment, the collapse temperature of an a4B7 antibody formulation is about -26°C. Without wanting to be bound by any particular theory, the greater the rate of rise, the greater the product's collapse temperature. The primary drying step can remove at least 50%, at least 60%, at least 70% or more of the solvent. In one aspect, the primary drying step removes more than 80°4 of the solvent from the anti-0.437 antibody formulation. Primary drying is dependent on the pratekira temperature and pressure. Conditions for primary drying can be determined empirically with different freeze drying and process parameters. Primary drying can even be mathematically modality based on product temperature. Mass and heat transfer equations (Milton, et al, (1997) PDA J qfPhurm Sci & Tech, 51: 7-16), coupled with knowledge of Rp and Kv, allow understanding of the combination and interaction of input variables including process input variables such as shelf temperature and pressure and formulation variables that are captured in the Rp value. These models can aid in determining the parameters to be used for an efficient process based on product temperature limitations by collapse temperature and equipment capacity.
dm A,(Q -E) ]rtP0= -6l44.96l!", + 24.0185 dt RU
Equation 1 Equation 2 t|=A,.K,.(IT,) dQ = AH dm dt ' dt Equation 3 Equation 4 5 Equation ] refers to the sublimation rate (dm/dt) during primary drying to the cross-sectional area container internal (Ap), ice vapor pressure (Po), chamber pressure (P,), and a standardized area of mass transfer resistance for the pie and lid (Rp). Po at the sublimation interface can be determined from equation 2, cinde Po is related to the temperature of the ice product at the sublimation interface, which is in 10 approximation from the product temperature (Tp), which can be measured with a thermocouple at the bottom of the bottle or can be derived from the above equations when the other variables are determined. Equation 3 refers to the rate of heat transfer from the shelf to the vials, where A, is the area of the E"flake, K, is the heat transfer coefficient of the vial, T, is the shelf temperature, and Tp is the product temperature Equation 4 couples the heat and mass transfer equations, where AH is the heat of sublimation. of product (TJ, chamber pressure (Pc), cake mass transfer resistance (Rp), and heat transfer coefficient (Kv) can affect the rate of sublimation.
An optional step after freezing and before primary drying is annealing. In this step the shelf temperature of the lyophilizer is raised above the glass transition of the formulation for a short period of time, for example, about 2 to 6 hours, about 3 to 5 hours, or about 4 hours, then the temperature of shelf is reduced again below the glass transition temperature of the formulation. Annealing can be used to crystallize r'olume agents and to form larger, more uniform ice crystals. The process of ar1e|aTT1Entc) can affect the constitution of terriPO because the dry rigged cake has a larger surface area than the dry uncurled cake. An annealing step of a «4B7 antibody formulation can be about 30°C to about 10°C or about 25°C to about -15°C. In one aspect, an annealing temperature for a formulation of antibody (14B7 is about -20°C. Secondary drying is generally conducted at a temperature that is above the freezing temperature of the liquid formulation.
For example, secondary drying can be conducted at about 10°C, about 20°C, about 30°C, about 40°C, or about 50°C.
In one aspect, the temperature for secondary drying is room temperature, eg 20-30°C.
The time for secondary sex:ageni should be sufficient to reduce the
5 amount of moisture at <5°4. In another aspect, the freeze-drying cycle includes freezing at about -45°C, annealing at about -20"C, re-freezing at about -45°C, primary drying at about -45°C at -24°C and 150 mTorr, and secondary drying at about 27°C and 150 mTorr.
Rp is affected by the solids content of the frozen DP and the DP thermal history (freeze, annular, and refreeze stages of 10) which affects the cake pore structure. The thermal history may further affect the secondary drying stage, where an area of wider surface may aid in water desorption (Pikal, et al. (1990) Int. J.
Pharm., 6 Ci: 203-217). Useful process parameters for control during the primary and secondary freeze-drying stages can be the shelf temperature and chamber pressure during each stage of the drying cycle.
To scale, freeze dryer load and solids content can affect the drying cycle.
The primary drying time can be affected by the solids content in the formulation.
At higher solids contents, for example, where general solids concentrations (excipients and/or protein) vary by more than 10 w/v% or more than 15 w/v°4, for example, 50 to 1OO'/ the variance of formulations whose drying time is determined, the drying time can be adjusted.
For example, a high solids formulation may have a longer drying time than a solids formulation.
In some modes, the percentage of freeze capacity usn dTyeT can range from CCTCà from 25 to about 100%. In °/, of higher capacity loading, the primary drying time may increase up to 2 times compared to 1 °/, of lower capacity loading.
The differences between the primary drying times in '/, of different load increase as the solids content increases.
In one embodiment, solids content is less than 20-25% and loading is 25-100%. The shank size can be selected based on the surface area that will be exposed to the shelf and freeze-drying during vacuum.
Drying time is directly proportional to cake height, so the jar size can be chosen based on what is determined to be a variable cake height.
A bottle with a large diameter to volume can provide a high amount of shelf contact for
" efficient heat transfer during the freeze-drying cycle.
A diluted antibody solution with a high volume of liquid will require more time to drain.
A balance in vial size versus formulation volume needs to be achieved, as larger vials can be more expensive to store and transport and have a greater proportion of formulation airspace, and can expose a high proportion of the formulation to the effects of degradation of moisture during long-term storage.
For a 300 mg dose, the Fasco tin of the anti-a4[37 antibody formulation may have a volume of 3 ml, 5 ml, 6 ml, 10 ml, 20 ml, 50 ml or 100 ml prior to lyophilization.
In one aspect, the vial size is 20 inl for a 60 mg/ml solution in a 300 mg dose.
After lyophilization, the vial can be sealed, eg resealed, in a vacuum.
Alternatively, a gas, eg dry air or nitrogen, may be allowed into the bottle before sealing.
Where oxidation is a concern, the gas allowed in the freeze-drying chamber may comprise a gas that retards or prevents oxidation of the freeze-dried product.
In one aspect the gas is an unoxygenated gas, eg nitrogen, or an inme gas, eg helium, neÔnium, argon, krypton or xenon.
In another aspect, the gas is nitrogen or argon.
In some embodiments, the volume of the anti-α437 antibody pre-lyophilization formulation is the same as the volume of reconstituted solution pre-administration.
For example, a formulation that is about 5.5 ml pre-lyophilization can be reconstituted to a volume of about 5.5 ml by adding an amount of liquid, for example water or saline, which occurs in solids volume accounting. dry.
In other embodiments, it may be desirable to lyophilize the anti-a4[37 antibody formulation in a different volume than the replenished solution vojume. example 0.25X, 0.5X, or 0.75X and reconstituted at 1X by adding less liquid, eg 75°4 less, half, or 25°4 less than the pre-lyophilization volume.
In one embodiment, a dose of 300 W' can be lyophilized as a 30 mg/ml antibody solution in 5% sucrose and reconstituted to a 60 mg/ml antibody solution in 10% sucrose.
Alternatively, the lyophilized anti-Q4lil7 antibody formulation can be reconstituted in a more dilute solution than the pre-lyophilized formulation.
Treatment with the Antibody Formulation In one aspect, the invention provides a method for treating a disease or disorder in a subject comprising administering to the subject the anti-α4,37 antibody formulation described herein in an amount effective to treat the disease or disorder, for example , in humans.
The human subject can be an adult (eg, 18 years of age or older), a teenager, or a child.
The human subject can be a person 65 years of age or older.
In contrast to alternative therapeutic dosing schedules, a human subject 65 years of age or older does not require any modification of the dosing regimen described herein, and may be administered the conventional anti-a4j37 antibody formulation described herein.
The subject may have had a lack of an adequate response with, loss of response to, or was intolerant of treatment with an immunomodulator, a TNF-α antagonist, or 10 combinations thereof.
The patient may have received prior treatment with at least one corticosteroid (eg, prednisone) for the inflammatory bowel disease.
An inadequate response to corticosteroids refers to signs and symptoms of persistently active disease despite a history of at least one 4-week induction regimen that included a dose equivalent to prednisone 30 mg daily orally for 2 weeks or intravenously pcir 1 week.
A loss of response to corticosteroids refers to two failed attempts to reduce corticosteroids below a dose equivalent to prednisone 10 mg daily by mouth.
Corticosteroid intolerance includes a history of Cushing's syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia, and/or infection.
An immunomodulator can be, for example, oral azathioprine, 6-mercaptopurine, or imctotrexate.
An inadequate response to an immunomodulator refers to signs and symptoms of persistently active disease despite a history of at least an 8-week regimen or oral azathioprine (>1.5 m£/kg), 6 mercaptopurine (>0.75 mg/kg), Oll n: ethotrexate (> 12.5 mE'/week). An immunomodulator intolerance includes, but is not limited to, nausea/vomiting, abdominal pain, pancreatitis, LFT abnormalities, lymphopenia, TPMT gene mutation and/or infection.
In one aspect, the subject may have had a lack of an adequate response with, loss of response to, or was intolerant of, treatment with a TNF-α antagonist.
A TNF-a antagonist is, for example, an agent that inhibits the biological activity of TNF-Cc, and preferentially binds to TNF-a, as a monoclonal antibody, for example, REM ICADE (infliximab), HUMIRA (adalimumab), CIMZIA (certolizumab pegol), SIMPONI (golimumab) or a receptor fusion protein such as ENBREL (etanercept). An inadequate response to a TNF-α antagonist refers to signs and symptoms of persistently active disease despite a history of at least a 4-week induction regimen of infliximab 5 mg/kg IV, 2 doses at least 2 distant sernanas; an 80 mg subcutaneous dose of adalimumab, followed by a 40 mg dose at least two weeks apart; or 400 mg subcutaneous cenolizumab pegol, 25 doses at least 2 weeks apart. A loss of response to a TNF-α antagonist refers to the recurrence of symptoms during maintenance dosing after prior clinical benefit. Inolerance of a TNF-cx antagonist includes, among others, infusion-related reaction, demyelination, congestive heart failure, and/or infection.
A loss of remission maintenance, as used herein for subjects with ulcerative colitis, refers to an increase in Mayo score of at least 3 points and a Modified Baron Score of at least 2. In another aspect, the present invention provides anti-a4B7 antibody formulations which (1) can bind α4f17 integrin in vitro and/or in vivo; and (2) can modulate an activity or function of an a4j37 integrin, such as (a) binding function (for example, the ability of a4j37 integrin to bind to MAdCAM (e.g., MAdCAM-1), fibronectin and/or VCAM -I) and/or (b) leukocyte infiltration function, including recruitment and/or accumulation of leukocytes and tissues (eg, the ability to inhibit lymphocyte migration to the tissue of the intestinal incusa). In one embodiment, an antibody in the formulation can bind an α4B7 integrin, and can inhibit the binding of α4B7 integrin to one or more of its ligands (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-I, fibronectin) , thereby inhibiting leukocyte infiltration of tissues (including recruiting:mto and/or leukocyte accumulation of tissues). In another embodiment, an antibody in the formulation can bind to an α4β37 integrin, and can selectively inhibit the binding of the α4β37 integrin to one or more of its ligands (e.g., MMCAM (e.g., MAdCAM-1), VCAM-I , fibroneciin), thereby inhibiting leukocyte infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues). Said anti-a4B7 antibody formulations can inhibit cell adhesion of cells containing an a4f17 integrin to vascular endothelial cells in mucosal tissues, including gut associated tissues, lymphoid organs or leukocytes (especially lymphocytes such as T or B cells) in vi/ ro and/or in vivo. In yet another embodiment, the anti-α4j37 antibody formulation of the present invention can inhibit the interaction of u4{37 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. In yet another embodiment, the anti-α4j37 antibody formulation of the present invention can inhibit the interaction of α4j37 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin
T_ 45/91 selectively, for example, without inhibiting the infection of o4B7 with VCAM.
The anti-q4B7 antibody formulations of the present invention can be used to modulate (e.g., inhibit (reduce or prevent)) the leukocyte (e.g., lymphocyte, monocyte) binding function and/or infiltration function of a4j37 integrin. For example, humanized immunoglobulins that inhibit the binding of α4f17 integrin to a ligand (i.e., one or more ligands) can be administered according to the method in the treatment of diseases associated with leukocyte infiltration (e.g., lymphocyte, monocyte) from tissues (including recruitment and/or accumulation of leukocytes in tissues), particularly from tissues expressing the MAdCAM molecule (eg, MAdCAM-1).
An effective amount of an anti-«437 antibody formulation of the present invention (i.e., one or more) is administered to a subject (e.g., a mammal, such as a human or other primate) to treat such a disease. For example, inflammatory diseases, including diseases that are associated with leukocyte infiltration of the gastrointestinal tract (including intestinal-associated endothelium), other mucosal tissues, or tissues that express the MAdCAM molecule (e.g., MAdCAM-l) (e.g., intestine-associated tissues such as small and large intestine lamina propria venules; and mammary gland (e.g., lactating mammary gland)) can be treated in accordance with the present method. Similarly, an individual having disease associated with leukocyte infiltration of tissues as a result of binding of 20 kukocytes to cells (eg, endothelial cells) expressing MAdCAM (eg, MAdCAM-l) can be treated in accordance with present inversion.
In one embodiment, diseases that can be treated in this way include inflammatory bowel disease (IBD) such as ulcerative colitis, Crohn's disease, ileitis, cdiac disease, non-tropical sprue, eneropathy associated with seronegative arthropathy, microscopic or collagenous colitis, eosinophilic gastroenteritis , or pouchitis resulting after protocolectomy, and ileoaRal anastomosis. Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Ukerative colitis can be moderately to severely active ulcerative colitis. Treatment can result in scarring of the mucosa in patients suffering from severely active to undecided ulcerative colitis. Treatment may also result in a reduction, elimination or reduction and elimination of corticosteroid use by the patient.
Insulin dependent diabetes mellitus and pancreatitis are other diseases that can be treated using the formulations of the invention. It has been reported that MAdCAM (for example,
MAdCAM-l) is expressed by some vessels in the exocrine pancreas of NOD (non-obese diabetic) mice, as well as BALB/c and SJL mice.
The expression of MAdCAM was reportedly induced in endothelium in inflamed islets of the pancreas of the NOD mouse, and MAdCAM was the predominant addressin expressed by endothelium of
5 islet of NOD in early stages of insulitis (Hanninen, A., et al., ./. C/in. /nves/., 92: 2509-2515 (1993)). Treatment of NOD mice with anti-MAdCAM antibodies
(e.g., anti MAdCAM-1) or anti f37 antibodies prevented the development of diabetes (Yang et al., Diabetes, 46:1542-1547 (1997)). In addition, accumulation of lymphocytes expressing cl4p7 within the islets fbi was observed, and MAdCAM-1 was involved in the attachment of lymphoma cells via a4B7 to inflamed islet vessels (Hanninen, A., et al., ./.C.C. /in. lnvesl., 92: 2509-2515 (1993)) or to the gastrointestinal tract in mantle cell lymphoma
(Geissmann et al., Am. J.
Pathol., 153:1701-1705 (1998)). Examples of inflammatory diseases associated with mucosal tissues that can be treated using a formulation of the invention include cholecystitis, cholangitis (Adams and
Eksteen Nature Reviews 6:244-251 (2006) Grant et al., Hepato/ogy 33:1065-1072 (2001)), for example, primary sclerosing cholangitis, Behcet's disease, for example, of the intestine, or pericolangile (duct biliary and surrounding liver tissue), and graft-versus-host disease (eg, in the gastrointestinal tract (eg, following a bone marrow transplant) (Petrovic et al.
B/ood 103:1542-1547 (2004)). As noted in the disease of
Crohn's inflammation usually extends beyond the mucosal surface, thus chronic inflammatory diseases such as sarcoidosis, chronic gastritis, eg autoimmune gastritis (Katakai et al., /n/, Immunol., 14:167-175 ((Katakai et al., /n/, Immunol., 14:167-175) 2002)) and other idiopathic conditions may be amenable to treatment. The invention further relates to a method for inhibiting leukocyte infiltration of mucosal tissue.
The invention further relates to a method of treating cancer (e.g. a cg4B7 positive tumor such as lymphoma). Other examples of inflammatory diseases associated with mucosal tissues that can be treated using a formulation of the invention include niastitis (mammary gland) and irritable bowel syndrome.
Diseases or pathogens whose etiologies exploit the interaction of MAdCAM (eg, MAdCAM-1) with ct4B7 can be treated with an anti-a4j37 antibody in a formulation described herein.
Examples of such diseases include immunodeficiency disorders, such as caused by human immunodeficiency virus (see, for example,
WO2008l40602).
A formulation of the invention is administered in an effective amount which inhibits the binding of α4,37 integrin to a NISM ligand.
For therapy, an effective amount will be sufficient to achieve the desired therapeutic (including prophylactic) effect (such as an amount sufficient to reduce or prevent α4,37 integrin-mediated binding and/or signaling, thereby inhibiting kukocyte adhesion and infiltration and/or responses associated cell phones). An effective amount of an anti-a±4j37 antibody, e.g., an effective titer sufficient to maintain saturation, e.g., neutralization, of «±4j37 integrin, can induce clinical response or remission to inflammatory bowel disease.
A formulation of the invention can be administered in a unit dose or multiple doses.
The dosage can be determined by methods known in the art and can be dependent, for example, on the individual's age, sensitivity, tolerance and general well-being.
Examples of modes of administration include topical routes such as nasal or inhalation or transdermal administration, enteral routes such as via a feeding tube or suppository, and parenteral routes such as intravenous, intramuscular, subcutaneous, intra-arterial administration,
intraperitoneal, or intravitreal.
Suitable dosages for antibodies may be from about 0.1 mg/kg body weight to about 10.0 mg/kg body weight per treatment, for example, about 2 mg/kg to about 7 mg/kg, about from 3 mg/kg to about 6 mg/kg, or from about 3.5 to about 5 mg/kg.
In particular embodiments, the dose administered is about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg.
The final dosage form, for example, after dilution of the reconstituted antibody (eg, in a saline or dextrose 5°/induction system) of anti-a4j37 antibody may be about 0.5 mg/ml at about 5 mg/ml for administration.
The final dosage form can be at a concentration of between about 1.0 mg/ml to about 1.4 mg/ml, about 1.0 mg/ml to about 1.3 mg/nil, about 1.0 mU/ml to about 1.2 mg/ml, about 1.0 to about 1.1 mg/nil, about 1.0 mg/ml to about 1.4 mg/ml, about from ],] mg/ml to about 1.3 mg/ml, about 1.1 mg/ml to about 1.2 mg/ml, about 1.2 mg/nil to about 1.4 mg µg/ml, about 1.2 mg/ml to about 1.3 mg/ml, or about 1.3 mg/ml to about 1.4 mg/ml.
The final dosage form can be at a concentration of about 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.1 mg/ml, about 1.2 nig/ml, about 1.3 mÊ/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.8 mg/ml or about 2, 0 mg/ml.
In one modality, the total dose is 180 mg.
In another method, the total dose is 300
"
A dose of 300 mg of anti-α4j37 antibody can be diluted in 250 µl saline or 5/4 dextrose solution for administration.
In some aspects, c) dosing schedule has two phases, an induction phase and a maintenance phase.
In the induction phase, the antibody or antigen-binding segment of the
It is administered in a route that rapidly delivers an effective amount of the antibody or antigen-binding fragment thereof suitable for certain purposes, such as inducing immune toxicity to the antibody or antigen-binding fragment thereof or for inducing a clinical response and alleviate the symptoms of inflammatory bowel disease.
A patient may be given a treatment in the induction phase when first 10 is being treated with an anti-a4B7 antibody, when being treated after a long absence from therapy, eg, more than three cases, more than four cases , more than six months, more than nine months, more than a year, more than eighteen months, or more than two years since anti-o4B7 antibody therapy or during the maintenance phase of antibody therapy anti-a4j37 if there is a return of symptoms of inflammatory bowel disease, for example, a recurrence of disease remission.
In some embodiments, the induction lysis regime results in a higher mean minimal concentration, for example, the concentration just before the next dose, than the mean steady-state minimal concentration maintained during the maintenance regimen.
In the maintenance step, the antibody or antigen-binding fragment thereof is administered in a manner that continues the response obtained by induction therapy with a stable level of antibody or antigen-binding fragment thereof.
A maintenance regimen can prevent the return of symptoms: or recurrence of inflammatory bowel disease.
A maintenance regimen can provide patient convenience, for example, be a simple dosing regimen or require infrequent excursions for treatment.
In some embodiments, the maintenance regimen may include administration of the anti-a4j37 antibody or antigen-binding fragment thereof, for example, in a formulation described herein, by a strategy selected from the group consisting of low dose, infrequent administration, self-administration and any combination of the above.
In one embodiment, for example, during an induction phase of therapy, the dosing regimen provides an effective amount of an anti-α4ji7 antibody or antigen-binding molecule in a formulation described herein to induce remission of an inflammatory bowel disease in a human patient.
In some embodiments, the effective amount of the anti-u4|37 antibody is sufficient to achieve from 5 µg/inl to about 60 µg/ml.
about 15 µg/ml to about 45 µg/ml, about 20 µg/ml to about 30 µg/ml, or about 25 µg/ml to about 35 µg/inl mean trough serum anti-antibody concentration. a4j37 by
5 end of induction phase.
The duration of the induction phase can be about four weeks, about five weeks, about six weeks, about seven weeks, or about Qito weeks of treatment.
In some modalities, the induction regimen may utilize a strategy selected from the group consisting of high dose, frequent administration, and a combination of high dose and frequent administration of anti-a4B7 aotibody or antigen-binding fragment thereof, for example, in a formulation described here.
The induction regimen may be once, or a plurality of more than one dose, for example, at least two doses.
During the induction phase, a dose may be given once a day, every other day, twice a week, once a week, every ten days, once every two weeks or every three weeks.
In some cases, induction doses are given within the first two weeks of therapy with the anti-a4B7 antibody. In one modality, induction regimen can be once at the start of treatment (day 0) and once about two weeks after starting treatment.
In another modality, the duration of the induction phase C is six weeks.
In another modality, the duration of the induction phase is six weeks and a plurality of induction doses are administered during the first two weeks.
In some modalities, for example, when initiating treatment of a patient with severe inflammatory bowel disease (eg, in patients who have failed anti-TNFm therapy, the induction phase needs to be longer than for patients with severe disease or moderate.
In some modalities, the induction phase for a patient with severe illness may be at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.
In one modality, an induction regimen for a patient with a severe illness may include a dose at week 0 (start of treatment), a dose at week 2, and a dose at week 6. In another modality, an induction regimen for a a patient with a severe disease may comprise a dose at week 0 (start of treatment), a dose at week 2, a dose at week 6, and a dose at week 10.
In one modality, for example, during a maintenance phase of therapy, the dosing regimen maintains a minimal serum concentration in the mean steady state,
.
for example, the plateau concentration immediately before the next dose, from about 5 to about 25 µg/ml, about 7 to about 20 µg/ml, about 5 to about 10 µg/ml, about 10 about 20 µg/ml, about 15 to about 25 µg/ml, or about 9 about 13 µg/ml of anti-a4B7 antibody. In another embodiment, the dosing regimen, for example, during a maintenance phase of therapy, maintains a mean steady state serum concentration of about 20 to about 30 µg/ml, about 20 to about 55 µg µg/ml, about 30 to about 45 µg/ml, about 45 to about 55 µg/ml, or about 35 to about 40 µg/ml of anti-a4|37 antibody. The dose can be given once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 10 weeks. A higher or more frequent dose, for example, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks may be useful to induce remission of active disease or to treat a new one. patient, for example, to induce tolerance to anti-ot4j37 antibody. A less flequent dose, eg once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks or once every 10 weeks, can be useful for preventive therapy, for example, to maintain remission in a patient with chronic disease. In one aspect, the treatment regimen is treatment on day 0, around week 2, around week 6, and every 4 or 8 weeks thereafter. In one modality, the maintenance regimen includes a dose every 8 weeks. In a mode where a patient on a dose every eight week maintenance regimen experiences a return of one or more symptoms of the disease, for example, has a relapse, the dosing sequence can be increased, for example, to once every 4 weeks. The dose can be administered to the patient in about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. The dosing regimen can be optimized to induce a clinical response and clinical remission in the patient's inflammatory bowel disease. In some modalities, the dosing regimen does not change the ratio of CD4 to CD8 in the cerebral spinal fluid of patients receiving treatment.
In some respects, a durable clinical remission, for example, a clinical remission that is sustained by at least two, at least three, at least four visits with a caregiver within a period of six months or one year after starting treatment, can be achieved with an optimized dosing regimen.
In some aspects, a durable clinical response, for example, a clinical response that is sustained for at least 6 inescs, at least 9 months, at least one year, after initiation of treatment, can be achieved with an optimized dosing regimen. In one embodiment, the dosing regimen comprises an initial dose of 300 mg, a second subsequent dose of 300 mg about two weeks after the initial dose, a third subsequent dose of 300 mg about six weeks after the initial dose. , followed by a fourth subsequent dose of 300 mg every four weeks or every eight sen weeks after the third subsequent dose.
In some modalities, the method of treatment, dose, or dose region 10 reduces the likelihood that a patient will develop an HAHA response to the anti-a4f17 antibody. The development of HAHA, for example, as measured by antibodies reactive to the anti-nt4j37 antibody, can increase the clearance of the anti-a4B7 antibody, e.g., reduce the serum concentration of the al1ti-a4B7 antibody, e.g., reduce the number of anti-antibody a4j37 bound to 44p7 integrin, thus making the treatment less effective. In some embodiments, to prevent HAHA, the patient can be treated with an induction regimen followed by a maintenance regimen. In some modalities, there is no break between the induction regime and the maintenance regime. In some embodiments, the induction regimen comprises administering a plurality of doses of anti-u4|37 antibody to the patient. To prevent HAHA, the patient can be treated with a starting dose of all, for example, at least 1.5 mg/kg, p at least 2 mg/kg, at least 2.5 mg/kg, at least 3 mg/kg , at least 5 mg/kg, at least 8 mg/kg, at least 10 mg/kg, or about 2 to about 6 mg/kg. once every two weeks or about every three weeks, of the standard dose when initiating therapy with an anti-a4j37 antibody. In some modalities, the method of treatment maintains at least 30°4, at least 40%, at least 5°/, at least 60%, at least 70%, at least 80°4, at least 90% or at least 95% of patients were HAHA-negative. In other modalities, the method of treatment maintains patients as HAHA-negative for at least 6 weeks, at least 10 weeks at least 15 weeks, at least six months, at least 1 year, at least 2 years, or for the duration of therapy . In some modalities, patients, or at least 30%, at least 40%, at least SO°/q, or at least 60% of patients who develop HAHA maintain a low titer, eg £125, of anti-a antibody. @7. In one modality, the treatment method maintains at least 7°/q of patients as HAHA-negative by pdo l _
r" 52/91 minus 12 weeks after initiation of therapy with an anti-ct4j37 antibody.
The formulation can be administered to an individual (eg, a human) alone or in conjunction with another agent. A formulation of the invention may be administered prior to, with or subsequent to administration of the additional agent. In one embodiment, more than one formulation that inhibits the binding of (x4B7 integrin to its ligand is administered. In said embodiment, an agent, e.g., a monoclonal antibody, such as an anti-MAdCAM (e.g., anti- MAdCAM-I) or an anti-VCAM-1 monoclonal antibody can be administered In another embodiment, the additional agent inhibits the binding of leukocytes to an endothelial ligand in a pathway other than the «4|37 pathway.
That said, an agent can inhibit the action, for example, of lymphocytes expressing chemokine receptor 9 (CC moiety) (CCR9) to thymus-expressed chemokine (TECK or CCL25) or an agent that prevents binding of LFA- I to the intercellular adhesion molecule (ICAM). For example, an anti-TECK or anti-CCR9 antibody or a small molecule CCR9 inhibitor, such as inhibitors disclosed in PCT publication WO03/099773 or WO04/046092, or an anti-ICAM-I antibody or an oligonucleotide that prevents expression of ICAM, is administered in addition to a formulation of the present invention. In yet another modality, an additional active ingredient (eg, an anti-inflammatory compound such as sulfasalazine, azathioprine, 6-mercaµtoµurine, 5-aminosa]icy]ic acid containing anti-inflammatory, another non-steroidal anti-inflammatory compound, a steroidal anti-inflammatory compound, or commonly administered antibiotics to control IBD (eg, cipmfbxacin, metronidazole), or other bilogical agent (eg, TNF alpha antagonists) may be administered in conjunction with a formulation of the present invention. In one embodiment, the dose of co-administered drug can be reduced over time during the treatment period by a formulation comprising the anti-cL4B7 antibody. For example, a patient being treated with a steroid (eg, prednisone, prednisolone) at the beginning of, or prior to, treatment with the anti-a4fl7 antibody formulation undergo a regimen of decreasing doses of steroid starting as early as 6 weeks those of treatment with the anti-«4P7 antibody formulation. The steroid dose will be reduced by about 25% within 4-8 weeks of onset of reduction, by 50 °/ by about 8-12 weeks and 75% by about 12-16 weeks of reduction during treatment with the anti-cL4B7 antibody formulation. In one aspect, at about 16-24 weeks of treatment with the anti-o4j37 antibody formulation, steroid dose may be
In another example, a patient being treated with an anti-inflammatory compound such as 6-mercaptopurine at the onset of, or prior to, treatment with the anti-Q4B7 antibody formulation could undergo a regimen of reduced doses of the anti-Q4B7 compound. -inflammatory similar to the reduction regimen for the dosage of steroid 5 as noted above. In one embodiment, the method comprises administering subcutaneously or administering via an effective amount of a formulation of the invention to a patient. , for example, dry, the process of administration may comprise a step of converting a formulation to a liquid state. In one aspect, a dry formulation may be reconstituted, for example, by a liquid as described above, for use in injection, for example, intravenous, intramuseular, or subcutaneous injection. In another aspect, a solid or dry formulation may be administered topically, for example, in a patch, cream, aerosol, or suppository. tory. The invention further relates to a method of treating a disease associated with leukocyte infiltration of tissues expressing the MAdCAM molecule (e.g., MAdCAM-1). The method comprises administering to a patient in need thereof an effective amount of an anti-α437 antibody formulation of the invention. In one embodiment, the disease is graft versus host disease. In some embodiments, the disease is a disease associated with leukocyte infiltration of tissues as a result of binding of leukocytes expressing a4B7 integrin to gut associated endothelium expressing the MAdCAM molecule (eg, MAdCAM-1). In other modalities, the disease is gastritis (eg, eosinophilic gastritis or autoimmune gastritis), pancreatitis, or insulin-dependent diabetes mellitus. Still in other modalities, the disease is cokcystitis, cholangitis, or pericolangitis.
The invention further relates to a method of treating inflammatory bowel disease in a patient. In one embodiment, the method comprises administering to the patient an effective amount of an anti-a4f17 antibody formulation of the invention. In some embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In other embodiments, the inflammatory bowel disease is celiac disease, enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, gastroenteritis (eg, eosinophilic gastroenteritis), or pouchitis. In some embodiments, treatment with an anti-c4j37 antibody does not alter the ratio of CD4:CD8 lymphocytes. CD4:CD8 ratios can be measured in blood,
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54/91
' lymph node aspirate, and cerebrospinal fluid (CSF). The ratios of CSF CD4+:CD8+ lymphocytes in healthy individuals are typically greater than or equal to about 1. (Svenningsson et al., J.
Neuroimmunol. 1995;63:39-46; Svenningsson et al., Ann Neurol. 1993; 34:155-161). An immunomodulator can change the ratio of CD4:CD8 to less than
5 that l.
Articles of Manufacture In another aspect, the invention is an article of manufacture which contains the pharmaceutical formulation of the present invention and provides instructions for its use.
The article of manufacture comprises a container.
Suitable containers include, for example, bottles, jars
]0 (eg, two-chambered vials, a liquid formulation vial with or without a needle, a solid formulation swab with or without a reconstitution liquid vial with or without a needle), syringes (such as two-chambered syringes , pre-filled syringes) and lesle tubes.
The container can be formed from a variety of materials such as glass, metal or plastic.
The container holds the formulation and a label on, or associated with, c)
15 container may indicate instructions for use.
In another embodiment, the formulation may be prepared for self-administration and/or contain instructions for self-administration.
In one aspect, the container holding the formulation may be a single-use vial.
In another aspect, the container holding the formulation may be a multi-use vial, which allows for repeated administration (for example, of 2-6 administrations) of the formulation, for example,
20 using more than one part of a reconstituted formulation.
The article of manufacture may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and inserts with instructions for use as noted in the above section, Clinical and Quality Analysis In another aspect, the invention is a method of determining that a pharmaceutical formulation meets product quality standards.
The method may comprise evaluating a lyophilized pharmaceutical formulation (eg humanized anti-cl4P7 antibody) comprising inspecting the formulation to assess appearance, determining reconstitution time, determining moisture content of lyophilized formulation, measuring
30 aggregated into the lyophilized formulation, measure fragmentation, measure oxidation and deamidation, and optionally evaluate biological activity and potency, whereby obtaining predetermined standards demonstrates that the product is suitable for clinical use.
Acceptable quality levels include £5.0% moisture, <40 minutes wash time.
' reconstitution, pH 6.3 ±0.3 of reconstituted liquid, 54.0 to 66.0 mg/ml antibody concentration, >55.0°4 major isoform per CEX, >96.0/4 monomer by SEC, £2.5% high molecular weight (aggregates), >90% H+L chains by SDS-PAGE, 60 140°4 of reference standard adhesion.
The invention will be further understood by reference to the following examples. It should not, however, be interpreted as limiting the scope of the invention. All literature and patent statements are incorporated herein by reference. DEVELOPMENT PROTOCOL FOR PREPARING THE
FORMULATION 10 An Anti-C437 Antibody Solution Bottles of high concentration anti-44B7 antibody congeal preparation (vedolizumab, 50 mM histidine, 125 mM arginine, 0.06-4 polysorbate 80, pH 6.3) are deconfused into room temperature for 16-24 hours. The thawed bottles are grouped in a stainless steel handling vessel and mixed. The preparation is then diluted with dilution buffer A (50 mM histidine, 125 mM art'inin, 0.06% polysorbal 80, pH 6.3) to 80 mg/ml vedolizumab and mixed. Sucrose is then added by diluting the preparation with dilution buffer B containing sucrose (50 mM histidine, 125 mM arginine, 40% sucrose, 0.06-4 polysorbate 80, pH 6.3). This step dilutes the an1i-cL4B7 antibody preparation to a liquid formulation of 60 mg/ml vedolizumab, 20 50 mM histidine, 125 mM arginine, 10°4 sucrose, 0.06°4 polysorbate 80, pH 6.3.
B. Lyophilization Liquid formulation of 60 mg/ml anti-α4[37] antibody in 50 mM histidine, 125 mM air, 0.06-4 polysorbate 80, 0.4 sucrose, at pH 6.3 is filled into vials 20 ml glass with 5.52 ml per vial and the caps are placed in the lyophilization position. The 25 vials are loaded onto shelves adjusted to about 20°C in a lyophilizer. After loading all the vials and closing the door, the shelf temperature is reduced until the solution freezes, around -45°C. After 3 hours at this temperature, the temperature of the shelves is raised to -20°C for annealing. After annealing for four hours, the temperature of the shelves is reduced to refreeze the solution, about -45°C.
30 After equilibrating the vials to this temperature, air is evacuated from the chamber. When the pressure is 150 tnTorr, the shelf temperature is raised to the primary drying temperature, about -24°C. The primary drying proceeds until all the crystalline ice has sublimed out of the jars. Then the shelf temperature is raised to 27°C for
"secondary drying for 16 hours, provided the humidity is approximately less than 2.5°4 of the freeze-dried formulation. When secondary drying is complete, nitrogen gas is filled back into the chamber until the ambient pressure is reached. jda The vials are closed and removed from the lyophilizer.
5 C. Storage and Use of Lyophilized Anti-u4F17 Antibody Lyophilized anti-o4B7 antibody vials are stored at -70°C, -20°C, 2-8°C or 25°C for desired periods of time. When ready to use, a bottle is equilibrated at room temperature. Then the contents of the vial are reconstituted with a syringe containing water for injection ("WFI") using a 21G syringe. The amount of WFI is determined so that the final volume of the reconstituted antibody solution is in the same volume as the pre-lyophilized solution. For a volume of 5.52 ml pre-lyophilization, 4.8 ml of WFI is added. The vial is gently shaken and then held for !0-30 minutes to allow the formulation to reconstitute, then the antibody solution is removed using a syringe and added to an IV bag for IV infusion to a patient.
EXEMPLIFICATION EXAMPLE 1 COMPARATIVE DATA FOR % BRANCH OF SUGAR AND AMINO ACIDS IN
LYOPHILLED FORMULATIONS A design experimental approach was carried out to observe the effect of changing the molar ratio of sugar (sucrose and mannitol) to protein, the molar ratio of arginine to protein, and the molar amount of histidine buffer. Histidine and arginine are known: they do not crystallize during the lyophilization process, making them cryo or lioprotective. 1.5 mL of formulation were filled into 5 mL lyophilized stalks with primary drying at -30°C, 150 mT and Secondary Drying at 20°C, 150 mT. Stability of reconstituted lyophilized formulations at 1.5 ml after conditions of Different storage is shown in Tables 1-3 (compiling 60 mg/ml results from two experiments) Figure 6A shows the predictive models for changes in percent monomer, percent aggregates, and percent major isoform when stored at 40°C when pH and molar ratio of sugar and arginine was varied. The stability of the formulation was better at low pH and high molar ratio of (sugar + arginine) to protein. In the n:tolar amounts of histidine evaluated, histidine did not affect stability of the formulation. All formulations had 1-2°4 moisture during storage.
57/9i Table 1: Percent change in monomer when stored at 5°C, 25°C/609/, RH, and 40°C/75% RH for 3 months Percent monomer was measured using size exclusion chromatography ( SEC).
Formulation '/, monomer by SEC j 60 ing/mL vedolizumab + | tO 5°C 25°C 40°C 3 mol 60%RH 75°4 RH) 3 mol 3 mol 25 mM histidine, 75 mM arginine, 2°4 I 98.1 98.1 97.8 96.5 I sucrose, 0.05% polysorbate 80, pH 6.3 II 25 mM histidine, 75 mM arginine, 4% I 98.0 98.2 98.0 97.5 I sucrose, 0.05% polysorbate 80, pH 6.9 II 50 mM histidine, 125 mM arginine, 2', I 98.0 98.3 98.1 97.4) sucrose, 0.05% polysorbate 80, pH 6.7 II 50 mM histidine, 125 mM arginine, 4° I 98.0 98.3 98.1 97.4 I sucrose, 0.05°4 polysorbate 80, pH 6.9 II 50 mM histidine, 125 mM arginine, 6°')) 98.7 98.4 98.4 98.1 sucrose, 1.5% mannitol, 0.06°4 ! ) polysorbate 80, pH 6.3 i 50 mM histidine, 125 mM arginine, 9%, i 98.7 98.3 98.1 98.3 l sucrose, 0.06°4 polysorbate 80, pH 6.3! Table 2: Change in percent water when stored at 5°C, 25°C/60% RH, and 40°C/75% RH for 3 months. Percent monomer was measured using tar-exclusion chromatography (SIiC ) [Formulation Aerated by 60 mg/ml. vedolizumab + I t 0 , 5°C i 25°C 40°C 3 mol I 60°4RH) 75% RH 3 mol 3 mol 25 mM histidine, 75 mM arginine, 2% 0.42 0.53 0.89 1.99 I sucrose, 0.05% polysorbate 80, pH 6.3 II 25 mM histidine, 75 mM arginine, 4% I 0.41 I 0.51 I 0.62 I 1.15 I sucrose, 0.05% polysorbate 80, pH 6.9 II 50 mM hislidine, 125 mM arginine, 2% I 0.42 I 0.47 I 0.60 I 1.23 • sucrose, 0.05% polysorbate 80, pH 6.7 I | 50 mM histidine, 125 mM arginine, 4% I 0.36 I 0.44 I 0.52 I 0.82 I sucrose, 0.05% polysorbate 80, pH 6.9 I I 50 mM histidine, 125 mM arginine, 6% I 0.53 I 0.49 I 0.51 I 0.56 I sucrose, 1.54 mannitol, 0.06% I: polysorbate 80, pH 6.3 I 50 mM histidine, 125 mM arginine, 9°4 I 0.51 I 0. 51 I 0.59 I 0.56) sucrose, 0.06% polysorbate 80, pH 6.3 I Table 3: Percent change of main isoform when stored at 5°C, 25"C/60°4 RH, and 40"C/75% RH for 3 months. Major isoform was measured using cation exchange chromatography (CEX).
FormulationQ main isoform per
60 mg/ml vedolizumab + t=o 5°C 25°C 40°C 3 mol 60%RH 7S°/o RH 3 mol 3 mol 25 mM histidine, 75 mM arginine, 2°/) 70.5 68, 8 67.4 66.3 sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 4°/, 70.8 98.9 68.0 67.7 sucrose, 0.05° 4 polysorbate 80, pH 6.9
. 50 mM histidine, 125 mM arginine, 2% 70.5 68.9 67.8 66.5 sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM arginine, 4°4 70.6 68 9.9 68.0 67.4 sucrose, 0.05% polyisosorbate 80, pH 6.9 50 mM histidine, 125 mM arginine, 6.4 69.6 69.5 69.3 67.4 sucrose, 1.5 % mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM arginine, 9°/0 69.5 69.3 69.2 68.1 sucrose, 0.06% polysorbate 80, pH 6.3
FIG. 6A shows the predictive models based on statistical analysis of 40°C data from Tables 1-3. The model for change in percent monomer per month at 40°C by SEC analysis is -3.10 + (0.386)*pH + 0.000516* ((moles of sugar + moles of
5 arginine)/moles of protein)). The model for allertion in percent aggregate per month at 40"C by SEC analysis is 2.43 - (0.263)*pH - 0.000787* ((sugar nioles +mole arginine)/mole protein)). for change is percentage of main isotome per month at 40°C by CEX analysis and -2.54 + (0.109)*pH - 0.001 30* ((moles sugar+moles arginine)/moles protein)). shows the results for the predictive models and the external lines show the confidence limit of 95'/, for the predictive models.
Fig. 6B shows alternative models based on the 40°C statistical analysis of Tables 1-3 when the input factors are pH, sugar:protein inolar ratio, and arginine:protein molar ratio.
The model for change in percentage of monomer per month to
40°C by SEC analysis is -3.02 + (0.370)*pH + 0.000482* ((sugar moks)/(moles of protein) + 0.000657* ((moles of arginine/moles of protein). The model for change in percentage of aggregate per month at 40"C by SEC analysis is 2.35 - (0.244)*pH - 0.000727*((moles of sugar)/(moles of protein) 0.00102* (( moles of arginine)/(moles of protein)) - The model for change in the percentage of major isoform per month at 40°C by CEX analysis is -2.92 + (0.210)*pH + 0.00164* ((moles of sugar) /)/(moles of protein) - 0.000220*((moles of arginine)/(moks of protein)) The center line shows the results for the predictive models and the outer lines show the confidence limit of 9S°/ q to
" predictive models.
Example 2 Stability Data · Three primary stability batches of the formulation (Lot A, B, and C) were tested for stability after storage at the prescribed storage condition (5 and 25°C/60°4 RH for up to 24 months) . All three Iotes contain the same liquid formulation as the lyophilized: 60 mg/ml anti-a4B7 antibody, 50 mM histidine, 125 mM arginine, 10°/, sucrose, 0.06°4 polysorbate 80, pH 6.3. For Lot A, 3.5 ml of the solution was filled into 20 ml vials and lyophilized, for Lots B and C, 5.52 ml of the solution was filled into 20 ml vials and lyophilized. In a separate study, a single drug formulation of 60 mg/ml anti-α4j37 antibody, 50 mM histidine, 125 mM arginine, 10°/, sucrose, 0.06% polysorbate 80, pH 6.3 was lyophilized in two volumes, 3.5 ml and 9.5 ml, respectively, to generate Lots R and S for stability samples, which were analyzed for 38 months. Whites are NT (not tested). The data (Tables 4-19) showed that the antibody formulations remained stable when stored for up to 38 months at 5°C and up to 30 months at 25°C/60°4 RH. All product attributes remained within specification until the time point of 38 mescs Tabda 4: Change in percent monomer by SEC when stored at :5°C.
Time Lot A Lot B Lot C Lot R Lot S (months' 0 99.8 99.8 99.8 98.9 98.8 l 99.8 99.1 99.2 98.8 99.2 3 99.8 99, 1 99.1 98.8 98.8 6 99.8 99.8 99.8 98.9 99.0 9 99.1 99.2 99.2 99.2 99.1 12 99.4 99.0 99.0 98.8 98.9 15 99.4 99.1 99,] 18 99.5 99.4 99.4 98.9 98.9 24 99.4 99.2 99.2 99.0 99.0 30 99.2 99.2 38 99.3 99.3 Table 5: Change in percentage of aggregates by SEC when stored at 5°C Time Batch A Batch C Batch R Batch S (months) 0 0.1 0.1 0.1 0.2 0.2 r 60/91
0,] 0.2 0.2 0.2 0.1 " r'I 3 0,! 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0, 2 I9 0.1 0.2 0.2 0.2 0.2 I 12 0.2 0.2 0.2 0.2 0.2 |15 0.2 0.2 0.2 I 18 0.2 0.2 0.2 0.2 0.2 I 24 0.2 0.2 0.2 0.2 0.2 I 30 0.2 0.2 I 38 0.2 0.2
Table 6: Percent change in main isoform by CEX when stored at 5°C.
Time Lot A Lot B Lot C I.lot R Lot S (levels) 0 68.6 69.9 69.5 72.7 71.6 1 67.5 68.9 68.8 71.2 72.0 3 68 .7 68.8 68.7 70.4 70.3 6 67.7 68.2 68.2 71.9 71.9 9 70.0 68.3 67.8 69.2 69.7 12 67.8 68.3 68.1 70.8 70.9 15 66.9 67.5 67.5 18 67.4 67.0 66.7 71.0 70.8 24 68.1 69.6 69.1 71, 3 70.9 30 68.5 68.6 38 73.6 73.1
5 Table 7: Change in percentage of acidic isoforms by CEX when stored at 5°C.
Time Lot A Lot B Lot C Lot R Lot S (months) 0 22.8 20.8 21.4 20.3 20.6 1 21.9 21.7 22.3 21.6 20.3 3 21.7 22.2 22.8 22.0 22.0 6 22.9 23.1 23.6 21.1 21.4 9 19.8 22.2 22.9 21.8 2!.8 12 22.9 21 .3 22.1 21.2 21.2 15 22.7 22.3 22.8 18 22.8 22.3 22.6 21.1 21.5 24 21.7 22.1 22.9 20.6 20.7 30 22.8 23.2 38 18.9 19.1
Table 8: Change in percentage of basic isoforms by CEX when stored at 5°C.
Time Lot A Lot B I.lot C Lot R Lot S (months) 0 8.5 9.3 9.1 8.1 7.8
"CJU) Table 9: Change in °/} (H+L) by reduced SDS Page when stored at 5"C.
/)) 1"table 10: Change in binding effectiveness when stored at 5°C.
)O) Table i 1: Change in °/, of humidity per KF when stored at 5°C Time |(months) I0 Il i3 i6 Lole A I 0,5 ) 0>5 ! 05 i 0.6 Lot B I cl6 ! 0.4 ! 0:6 i 0.7 Lot C ) 0.6 " I 0.6 I 0.6 l 0.5 Lot R l 0.8 j 0.8 |i,o Lot S I 1.3
12 0.6 0.6 0.7 0.9 0.9 24 0.5 0.7 0.7 0.9 0.9 30 0.7 0.7 Table 12: Change in percentage of monomer by SEC when stored at 25°C/60%RH, Time Batch A Batch B Batch C Batch RI BatchS (months) 0 99.8 99.8 99.8 98.9 98.8 l 99.8 99.1 99.2 98 ,7 98.7 3 99.8 99.0 99.0 98.6 98.5 6 99.8 99.7 99.7 98.9 98.9 9 99.0 99.1 99.1 99.1 99.1 12 99.3 98.9 98.9 98.8 98.9 15 99.3 99.0 99.0 18 99.4 99.3 99.3 98.7 98.9 24 99.2 99.1 99.1 98.9 98.9 30 99.0 99.0 Tabkla 13: Change in percentage of aggregates by SEC when stored at 25°C/60%RH I Tenipo ! LotA I Lot8 I LotC I LotR I Lot S 6))))/ Table 14: Change in percentage of main isoform by CEX when stored at 25"C/60°4RH.
Time Lot A Lot B Lot C Lot R Lot S (months) 0 68.6 69.9 69.5 71.7 71.6 ! 67.2 68.4 68.6 71.0 3 68.1 68.6 68.2 70.3 70.3 6 65.9 67.8 67.8 71.5 71.1 9 69, 3 67.5 66.3 68.6 69.0 12 66.7 67.5 67.4 70.1 70.2 15 66.2 66.6 66.8 18 66.1 65.8 64.9 70 .0 70.3 24 66.7 68.4 68.2 70.6 70.1 30 67.2 67.2
Table 15: Percent change of acidic isoforms by CFX when stored at
25'C/60°4RH,
Time Lot A Lole B I-lot C Lotc R Lot S (months) 0 22.8 20.8 21.4 20.3 20.6 1 21.9 21.8 22.2 21.4 21.6 3 21 .7 22.2 22.8 21.8 22.0 6 22.6 22.9 23.5 21.1 21.4 9 19.9 22.1 23.1 2],8 21.8 12 23, 0 21.4 22.0 21.3 2!.3 15 22.5 22.1 22.7 18 22.6 22.1 22.6 21.3 21.5 24 21.7 21.9 22.6 20.7 20.7 30 22.7 23.2 Table 16: Change in percentage of basic isoforms by CEX when stored a
25°C/60%RH.
Time Lot A Lot B Lot C Lot R Lot S (mcses) 0 8.5 9.3 9.1 8.1 7.8 l 10.8 9.8 9.2 7.4 7.3 3 10.3 9.3 9.0 7.8 7.7 6 11.5 9.3 &7 7.4 7.5 9 10.8 10.4 10.6 9.7 9.3 12 10.3 11.1 10.7 8.7 8.5 15 11.3 11.2 10.6 18 !1.2 12.1 12.5 8.7 8.2 24 !1.6 9.7 9.1 8.7 9.2 30 10.2 9.6 Table 17: Change in °/, (H+L) by SDS Page reduced when stored at 25°C/60%RH
Time Lot A Lot B Lot C Lot R Lole S !(months)
I 0 98 98 98 I98 98 98 _J I 98 I 98 j 96 i 96 97 98 98 98 98 I6 ! 97 I 97 I 97 I 97 I 97 97 97 97 98 98 L12 98 96 96 98 98 I 15 . ! 97 I 97 I 97 |'8) 98 I 97 |97 j99 I 24) 98 I 97 I 98 !99 99 | 30 I 97 I 98 Table 18: Change in binding efficacy when stored at 25°C/60°4RH. rrempoo'eÃ"TTo'eoteo'eWTTo'e|
(months) 0 107 106 105 93 102 1 llS 103 109 3 92 ll3 100 96 94 6 109 89 97 10l 114 9 97 89 85 97 102 12 83 91 ]23 15 96 91 96 18 106 123 87 92 102 24 103 82 90 98 94 30 84 114 Tabda 19: Change in °/) of humidity per KF when stored at 25'C/60°/,RH I Weather ! Lot,4 I Lot8 | Lotc I LotR I Lot S I (months) Õ°°°'7° U)7OU|) I 30 I ! 0.8 ! 0.7 Cation Exchange Chromatography (CEX) A phosphate/sodium chloride gradient on a ffac cation exchange column is used in a high performance liquid chromatography system to separate charged species in anti-ct4B7 antibody formulations and determine the loading composition of the antibody species. Acidic isoforms elute before the main isoform and basic isoforms elute after the main isoform.
Stability data for all vedolizumab lots generated using a CEX assay are presented in Tables 3, 6-8 and 14-16. The Tables show that under these 10 storage conditions, there was no trend of reduction of °/) Main Isoform below 55.0%.
Size Exclusion Chromatography (SEC) SEC is performed using a SEC analytical column (Tosoh Bioscience, LLC, King of Prussia, PA) The mobile phase is a saline solution buffered in phosiatin and the absorbance is monitored at 280 nm.
Stability data generated using an SEC assay is presented in Tables 1, 2, 4, 5, 12, and 13. The Tables show that none of the storage conditions listed resulted in a reduction of the monomer °/o below 96.0% . Similarly, the % of Aggregates remained <2.5% for all batches in all
65/91'. storage conditions listed.
SDS-PAGE Assay SDS-PAGE is performed using an Invitrogen (Carlsbad, CA) Tris-Glycine gel, 4-20% for reducing condition and 4-12°4 for non-reducing condition. The reconstituted antibody formulation is diluted in Lampão liquid formulation, then diluted one by two with Tris-Glycine SDS sample buffer (2X Sample, Invitrogen) with 10°/ü 2-mercaptoethanol (reducing sample buffer) or without 2-mercaptoethanol (non-reducing sample buffer). Samples are briefly heated and loaded against a high molecular weight marker (Invitrogen). The gels are stained with colloidal Coomassie blue 10 (Invitrogen) according to the manufacturer's instructions. Protein bands are analyzed by densilometry to identify c) °/) of heavy chain and light chain and °/, IgG for non-reduced E'els.
Stability data generated using a reduced SDS-PAGE assay is presented in Tables 9 and 17. No notable change was observed for the 15 chain Heavy + Light (H+L) under all storage conditions for all lots of stability. The banding pattern was similar to the reference standard and the °/) (H+L) remained at a level >90%, Binding Efficiency Hu'l" Cells 78 (Human T cell lymphoma cells, American Type Culture 20 Collection, Manassas, VA) suspended in 1°4 BSA in PBS, 0.01°4 sodium azide is treated with serial dilutions of primary test antibody. After incubation on ice, cells are washed and treated with fluorescently labeled secondary antibody After another wash, the cells are fixed and suspended in FACS reagent for analysis by flow cytometry (Becton Dickinson Franklin Lakes, Nj), see also US Patent 7,147,851.
25 Binding efficacy of vedolizumab was measured relative to the reference standard and reported as °/) of reference standard at EC50. Stability data are shown in Tables 10 and 18. The data for the reherence pattern % showed variability but remained within specification limits under all storage conditions. No batch of vedolizumab evaluated showed a trend of reduced binding efficacy under the listed storage conditions.
Karl Fischer Moisture The formulation is titrated with methanol for a Karl Fischer coulometric moisture determination. Humidity data are presented in Tables ll and 19.
.
" All batches evaluated had less than 5°/) moisture in all storage conditions listed.
Epilar isoelectric focusing (cjEF) cIEF is performed using a complete 1CE280 column in CIEF 5 detection system (Convergént Biosciences, 'J'oronto, Ontario). The choice of ampholyte can be recommended by the manufacturer or it can be a combination of commercially available ampholytes. A useful combination is a mixture of 3-10 and 5-8 PHARMALYTE'" (GE Healthcare, Piscataway, NJ).
Example 3: Staging Modeling of the freeze-drying process Quality by design was used while manipulating the charge in the freeze-dryer and the solids content of the emulsion. The load was varied from 33 to 100'/. A formulation solids content was varied from 9 to 27% including in the fillers a formulation that was 0.5X, 1.0x and 1.5X of the target formulation. These settings had similar Tg'. With higher °4 solids, the primary drying time is increased. In addition, the higher solids color, the product temperature increased due to the higher Rp. The load still had an effect on both stages of drying (FIG. 8).
Example 4: Non-Clinical Safety Study A study has been designed to compare the effect of natalizuniab and vedolizumab on CNS immune survival in Rhesus EAE. Eight animals were dosed with a placebo conrol once a week. Seven animals were dosed at 30 mg/kg, once per serjana, conjo naEa]1LuTIlabe Seven animals were dosed at 30 mg/kg, once a week, conjo vedolizumab. Clinical symptoms of EAE were noted; the frequency and proportion of leukocyte subsets in CSF were measured by flow cytometry; the total T2 lesion burden in the brain was measured using MRI; and brain lesion burden and demyelination was measured using histopathology.
Vedolizumab did not delay the onset of clinical symptoms of EAE compared to pIacebo ccntrole. This did not inhibit the incidence of EAE, nor the magnitude of clinical scores.
Natalizumab significantly (p<0.05) reduced the onset of cfinic symptoms of EAE compared to placebo control. This inhibited the incidence of EAE, the magnitude of clinical scores. (Fig. 9) Vedolizumab did not prevent CSF infiltration by leukocytes, T lymphocytes (T helper lymphocytes, cytotoxic T lymphocytes), B lymphocytes, natural killer cells, or monocytes. In contrast, natalizumab inhibited CSF infiltration.
J 67/91 Vedolizumab did not inhibit the accumulation of brain lesions, as detected by increased T2 and reduced MTR values via MRI. Natalizumab prevented lesion formation in all but one animal. Significant inhibition (p<0.05) in brain infiltrates and demyelination was measured by histology.
5 The ct4(37 integrin was saturated by vedolizumab during the investigation, as shown by a competitive binding assay between vedolizumab dosed in vivo and in an analytical monoclonal anti-a4j37 antibody added ex vivo. The anti-q4l37 analytical mAb does not bind to memory T helper lymphocytes in animals dosed with vedolizumab. The absence of the effects of vedolizumab on CNS is therefore due to the gastrointestinal tropic biology of Cl4P7 integrin. In summary, vedotizumab (a c4j37 antagonist) does not inhibit EAE. In contrast, natalizumab (antagonist cl4B1 and a4fl7) inhibits EAE. α4f11 integrin mediates CNS infiltration into EAE. Thus, vedolizumab may have a lower risk of patients predisposed to PML than natalizumab because it does not antagonize ct4|3l integrin and confer survival 15 ijnune CNS and jn EAE Rhesus. Example 5: Phase I Clinical Trial with Vedolizumab Forty-nine healthy subjects were randomized and received a single dose of study medication: 39 subjects received vedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125 mM chloride). sodium, 0.05°4 polysorbate 80, pH 6.0 20 (long term storage -70°C and up to 3 months at -20°C)) and 10 subjects received placebo. Of the 39 subjects who received vedolizumab, 8 subjects each received a dose at 0.2, 2.0, 6.0, and 10.0 mg/kg and 7 subjects received vedolizumab at 0.5 mg/kg. All 49 subjects completed the study. There were no notable differences across vedolizumab cohorts for any demographic or baseline characteristics. Mean age ranged from 35.4 to 51.0 years; the ages of the individual subjects ranged from 21 to 63 years.
Results PK Vedolizumab was administered as a 30-minute intravenous infusion at 0.2 to 10.0 mg/kg. Cmax and area under the curve of serum drug concentration-time values (AUC) increased with c) dose increase. Dose-corrected Cmax was approximately the same across cohorts, indicating dose proportionality for this parameter. The dose-normalized area under the serum drug concentration value from zero to infinity (AUCO-,ní) increased with increasing dose up to 2.0 mg/kf', indicating that there was a non-linear increase in A[JC0 mfgom the increasing dose over the lower range of doses administered in this study. Then, AUC0-inf increased proportionally with dose, indicating linearity of AUC0-,nf over the 2.0 to 10.0 mg/kg dose range. The increase in AUCo,nt'fDi is approximately 2.4 times greater than expected at the 10.0 5 nig/kg dose compared to the 0.2 mg/kg dose.
Similarly, estimates of clearance, volume of distribution, and terminal half-life were dose-dependent over the 0.2 to 2.0 mg/kg dose range. As the dose increased, clearance was reduced, the volume of distribution increased and, consequently, the terminal elimination half-life was prolonged. However, from 2.0 to 10.0 mg/kg, there was no apparent change in these parameters, suggesting a saturation of a rapid elimination process for vedolizumab at low concentrations. Slower linear dimination processes possibly account for a large fraction of vedolizumab clearance at higher doses.
In some subjects who developed HAHA to vedolizumab, faster clearance of vedolizumab was observed compared to HAHA-negative subjects within the respective dose level.
Table 20: Vedolizumab PK overview by dose cohort after IV administration of 0.2-10.0 mg/kg vedolizumab in healthy subjects (PK analysis set) Parameter VDZ N Mean SD Mean % CV Median Min Max geometric dose Cmax (µg/ml) 0.2 4 5.65 0.629 5.62 11.1 5.45 5.13 6.56 mg/kg 0.5 4 10.6 2.09 10.4 19.7 10, 6 8.07 13.1 mg/kg 2.0 7 59.3 11.6 58.4 19.6 58.4 - 47.6 78.4 mg/kg 6.0 6 151 19.1 150 12, 6 157 120 168 mg/kg 10.0 7 243 22.1 243 9.07 242 213 281 mg/kg AUC &ü,,, 0.2 4 31.6 4.98 31.3 15.8 31.6 25 .7 37.5 (da"µg/mL) mg/kg 0.5 4 127 48.0 119 37.9 129 70.9 178 I mg/kg
69/9!
I 2.0 7 964 147 955 15.2 972 772 117C I mg/kg
I 6.0 6 3090 749 3020 24.2 2830 2360 4100 I mg/kg
I 10.0 7 4870 624 4840 12.8 4750 4120 5870 I mg/kg
Â(JCO-nf ) 0.2 4 39.5 5.79 39.1 14.7 40.2 31.7 45.7 (dia*µg/ml) ! mg/kg i 05 4 134 48.9 127 3€5 134 79.2 188 I mg/kg 7 979 146 969 14 9 993 784 1180 ! mg/kg µr_ 6 3100 750 3030 24.2 2840 2390 4110 i mg/kg µir 7 4880 637 4850 13.0 4750 4130 5920 l mg/kg
Vz(L) | 0.2 4 4.02 0.151 4.02 3.76 4.03 3.83 4.18 l mg/kg
I 0.5 12.€ 4.52 5.84 4 4.92 0.620 4.89 4.€6 I mg/kg
I 2.0 19.9 2.29 4.27 7 3.34 0.665 3.28 3.23 I mg/kg
6.0 6 2.98 0.644 2.92 21.6 2.98 2.06 3.98 I mg/kg
) 10.0 7 2.89 1.02 2.73 35.2 2.98 1.49 4.58 I mg/kg
LC (L/day) I 0.2 4 0.413 0.042 0.412 10.1 0.395 0.388 0.476 I mg/kg I 0.5 0.212 0446 4 0.310 0.106 0.297 34.3 0.291 : mg/kg m 7 0.165 0.018 0 164 10, 7 0.162 0.145 0.194 I mg/kg i 6.0 6 0 140 0.031 0.136 22.0 0.145 0.083 0.166 I mg/kg
I 10.0 7 0 140 0.024 0.139 16.9 0.135 0.103 0.171 I mg/kg txz (da 0.2 4 6.79 0.736 6.76 10.8 6.95 5.79 7.47 I mg/kg
I 0.5 4 11.7 2.83 11.4 24.2 11.4 9.09 14.8 mg/kg 2.0 7 14.1 2.67 13.9 18.9 14.3 10, 6 17.5 mg/kg 60 6 15.1 3.15 14.8 20.9 14.0 11.9 20.3 mg/kg 10.0 7 14.8 7 38 13.7 49.8 12, 5 8.26 30.7 mg/kg Abbreviations: AUCò ,nf--area under the drug concentration-time curve, extrapolated to infinity; AUC0-,|a,t= area under the drug concentration-time curve from the time of administration to the last measurement time point where the concentration is above the lower limit of quantification; CL total clearance; Cn,aK=maximum drug 5 concentration; tjQ terminal half-life; V, volume of distribution based on terminal phase.
After reaching the serum C,,,,,,, concentrations of Vedolizumab fall in a monogeneral moo exponentially until C concentrations reach approximately 1 to 10 mg/L. Then the concentrations appeared to be in a non-linear fashion.
The values of Cm, and AUC increased with increasing dose. For the 10 data available, the dcise-corrected Cmax was approximately the same across the cohorts, indicating dose proportionality for this parameter. AUCV-,,,f normalized per dose with increasing dose up to 2.0 mg/kg, indicating that there was a non-linear increase in AUCQjnf with increasing dose over the lower range of doses administered in this study. Then, ÀÜCOmf increased the proportionality with dose, indicating linearity of lS AUC0-,,,f over the dose range 2.0 to 10.0 nig/kg. The increase in AUC+i,,r was approximately 2.4-fold greater than expected at the 10.0 mg/kg dose compared to the 0.2 ing/kg dose. PD Results Vedolizumab PD parameters after a 30-minute intravenous infusion of 0.2 to I 0.0 mg/kÈ vedolizumab per cohort are summarized in Table 21 and Table 22 for ACÉ-I and MAdCAM respectively.
Table -2 1: Overview of pharmacodynamics of vedolizumab, percent inhibition of °/oACt l" [CD4" CD45RO'l'°], by dose cohort after IV administration of 0.2-10.0 mg/kg vedolizumab in healthy subjects (PD analysis set) Parameter VDZ N Mean SD Mean "µct I dose I l II geometric I Emu(%njbitionQ) I 0.2 I4 i 99.6 |0.387 j99.6 jc.388 j99.6 i 99.1 i 100 mg/kg
0.5 4 99.5 0.599 99.5 0.602 99.5 989 100 mg/kg
2.0 6 99.9 0.172 99.9 0.172 100 99.6 100 mg/kg
6.0 6 100 0.000 100 0.000 100 100 100 mg/kg
10.0 6 99.7 0.326 99.7 0.327 99.8 99.3 100
'mg/kg
AUECeim 02 4 4030 1010 3920 25.2 4090 2760 5160
(%inhibition "d) mg/kg
0.5 4 6430 1450 6300 22.6 6530 4860 7810 mg/kg
2.0 6 13200 623 13200 4.72 12900 12800 14500 mg/kg
6.0 6 16700 3030 16500 18.1 16300 13300 20100 mg/kg
10.0 6 19300 644 19300 3 33 19600 18200 199000 mg/kg
AUEC0-,ntArea under drug effect curve versus time from time 0 to the time of the last non-zero concentration; E,nax= maximum drug effect
Table 22: E'eral view of pharmacodynamics of vedolizumab, percentage inhibition of
%MADCAM"[CD4"CD45RO"°], per dose line after IV administration of 0.2]0.0 m£'/kg vedolizumab in healthy subjects (PD analysis set)
Parameter VDZ N Mean SD Mean '/oCl Median Min Max geometric dose
Emu (% nibition) 0.2 4 99.2 0.537 99.2 0.542 99.4 98.4 99.6 mg/kg
0.5 4 996 0.323 99.6 0.324 99.5 99.3 100 mg/kg
2.0 Eil 997 0.365 99.7 0.366 997 99.2 100 mg/kg
6.0 6 99.8 0.279 99.8 0.280 100 99.4 100 mg/kg
10.0 $100 0.000 100 0.000 100 100 100 mg/kg
AUEC±m 0.2 4 4000 576 3970 14.4 4210 3160 4440
(%in'bition"d) mg/kg
I 0.5 |4)6770 |1400 I 6660 I 20.6 I 6840 I 5170 I 8230 mg/kg 2.0 6 13000 796 13000 6.12 13000 11700 13900 I 3320 I 15900 ) 20.5 I 15800 j11800 j20000 I mg/kg 10.0 6 17700 1330 17700 7.5 17700 16500 19000 mg/kg AUECo ,nr-ar¢a under drug effect curve versus time of time 0 to the time of the last non-zero concentration; Ema = maximal drug effect.
Vcdolizumab inhibited PD, Act-1 and MAdCAM-1-Fc parameters almost maximally at all time points where vedolizumab was measured in serum. Once the 5 concentrations of vedolizumab dropped below the detection limit of the assay, inhibition of Act-1 and MAdCAM-I-Fc returned to approximately the basal level.
In some subjects who developed HAHA to vedolizumab, a more rapid loss of g4B7 receptor saturation was observed compared to HAHA negative subjects within the respective dose level.
Safety results Vedolizumab was generally safe and well tolerated in single IV doses up to 10.0 mg/kg'. No deaths, serious adverse events (SAEs) or AEs leading to study discontinuation occurred during the study.
immunogenicity / Human Anti-Human Antibody Formation (HAHA) ()it1 subject (iO"/j) ril) p]acEbU group and 2] (54"4i) subjects il()s combined dose groups of vedolizumab had a HAHA positive at some point during the study.
Although HAHA positive samples were observed in all dose cohorts, FJAHA titers >125 were found only in the 2 lowest dose groups of vedolizumab. Dose-dependent suppression of HAHA formation has been previously observed with vedolizumab. Nineteen of the 22 vedolizumab-treated subjects who were HAHA positive had neutralizing HAHA present. ' 1" Table 23: Overview of Human Anti-Human Antibody Findings: Safety Population Placebo 0.2 0.5 2.0 6.0 10.0 VDZ jN 10 I mg/kg ; mg/kg I mg/kg I mt'/kg I mg/kg I combined I VDZ ) VDZ i VDZ I VDZ I VDZ IN 39
IN8! N=7 I N=8 ! N=8 IN8 Subjects 10 8 7 8 8 8 39 I tested Any 1 (10) . 6 (75) 4 (57) 2 (25) 3 (38) 6 (75) 21 (54)
I HAHA positive, n (O/q) Title plus ] (10) 4 (50) 2 (29) 2 (25) 3 (38) 6 (75) 17 (44) I allo of l
I HAHA <125, n(°/o) Title plus 0 2 (25) 2 (29) 0 0 0 4 (10) I high of I
I HAHA >125, n(%) Any 0 5 (63) 4 (57) 2 (25) 3 (38) 5 (63) 19 (49)
I HAHA neutralizing positwo, n(%) Title plus 0 3 (38) 2 (29) 2 (25) 3 (38) 5 (63) 15 (38) high of
HAHA neutralizing <125, n(°/o) Title plus 0 2 (25) 2 (29) 0 0 0 4 (10) high of
I HAHA neutralizing Z125, n(°/,) One subject in the placebo group and 11 subjects in the vedolizumab group were persistently positive for HAHA.
Table 24: Overview of human anti-human antibody status (safety population) Placebo 0.2 0.5 2.0 6.0 10.0 VDZ N 10 mg/kg mg/k£' mg/kg mg/ kg mg/kg combined VDZ VDZ VDZ VDZ VDZ N-39 N=8 N 7 N=8 N8 N8 HAHA 9 (90) 2 (25) 3 (43) 6 (75) 5 (63) 2 (25) 18 ( 46) negative' n(%) HAHA' 0 2 (25) l (14) 1 (13) |1(13) 5 (63) ) 10 (26) isolated n(%) HAHA' l (10) i 4 (50) )3(43) ! t(13) I 2(25) 1 (13) l 11(28) persistent n(%) a HAHA Negative: Subjects with no positive HAHA results b HAHA isolated: Subjects with only 1 HAHA positive sample with titer <25 c HAHA Persisteritis: Subjects as being 2 or more HAHA positive samples, or 1 positive sample with titer >25 Conclusions This phase 1 study characterized the PK/PD and baseline profiles of CHO cell-derived vedolizumab. The results of this study were used to support 10 dose selection for pivotal phase 3 studies in inflammatory bowel disease. Vedolizumab demonstrated dose proportionality over the dose range tested for the Cmax parameter; however, dose-dependent changes in AUCO-inf, CL, Vz, and t1/2 were observed from 0.2 to 2.0 mg/kg, suggesting non-linear PK behavior of vedolizumab. At dose levels greater than 2.0 mg/kg, no other changes in these parameters were observed, suggesting a rapid process of elimination for vedolizumab at low concentrations. Slower elimination processes possibly account for a large fraction of vedolizumab clearance at higher doses. Vedolizumab inhibited PD, Act-1 and MAdCAM-1-Fc parameters, at or near maximum levels at all time points when vedolizumab was measured in serum.
Once vedolizumab concentrations fell below the assay's detection limit, the
Inhibition of Act-l and MAdCAM-I-Fc returned to approximately baseline level. In some subjects who developed HAHA to vedolizumab, faster clearance of vedolizumab and loss of ct4j37 receptor saturation was observed compared to HAHA-negative subjects within of the respective dose level.
5 Vedolizumab was well tolerated. No deaths, SAEs, or AES leading to discontinuation of study drug administration occurred during the study, nor was any relationship of dose and toxicity observed. No systemic opportunistic infections (including PML) or neoplasms have been reported. Unlike non-specific α4 antagonists, vedolizumab was not associated with 10 lymphocytosis or mild increases in circulating eosinocytes, basophils, or monocytes, nor was there any evidence of lintocyte depletion. Vedolizumab did not induce HAHA formation, but the highest titers (>125) were only seen in the 2 lowest dose groups, a finding that supports the observations of a dose-dependent reduction in immunogenicity. These data 15 showed that the administration of higher doses of vedolizumab can minimize the clinically significant formation of HAHA. In conclusion, vedolizumab was generally safe and well tolerated when administered in single doses of 0.2 to 10.0 mg/ky to healthy subjects. Example 6: Determination of vedolizumab at CD4:CD8 ratio Healthy subjects ages 18-45 were treated with a single dose of 450 mg vcdolizumab reconstituted from a lyophilized formulation of 10% sucrose and diluted in a 0.9% infusion set saline. Cerebrospinal fluid (CSF) was collected by IOrnb:ir puncture before (basal) and 5 serrian punctures after a single 450iny dose of vedolizunwbe. Each subject served as its own control. 25 A 5-week time point was selected based on a previous study that showed that MS patients treated with natalizumab demonstrated effects on CSF CD4+:CD8+ lymphocyte ratio and reduction in number of brain lesions after only one dose (Stuve et al. Arch Neurol, 2006;63:1383-1387; Stuve et al. Ann Neurol. 2006;59:743-747. Milkr et al. N Engl J Med. 2003;348(1):15-23); and because at 5-30 weeks, a dose of 450 mg of vedolizumab is sufficient to saturate the target and provides serum concentrations that exceed the minimum levels in the estimated steady state associated with phase 3 of the 300 mg every 4 dose regimen weeks. Approximately 15 ml of CSF was obtained from each subject for immunophenotyping. CSF samples are included for analysis if they meet the following criteria: <10 RBCs/µL per sample (to minimize peripheral blood contamination); negative result for CSF culture; adequate T lymphocyte number in each flow cytometry sample; and no detection of serum antibodies to vedolizumab.
5 Median at week 5 (34.80 µg/mL) and individual subjects' serum vedolizumab concentrations (range 24.9-47.9 µg/mL) were greater than the minimum steady-state concentration (-24 µg/ mL) for the phase 3 dose regimen. A high degree (>90%) of α7 receptor saturation was observed at week 5 as measured by MAdCAM-I-Fc, indicating its target's vedolizumab saturation at time of the evaluation of the 10th point of evaluation.
Vedolizumab was not detected in any CSF sample (detection limit = 0.125 µg/mL).
Effect on CD4+ and CD8+ T lymphocyte numbers and ratio Vedolizumab did not significantly reduce the CD4+:CD8+ ratio (Table 25). None of the subjects had a ratio after the CD4+:CD8+ dose <1 (p < 0.0001 (side t test)). Vedolizumab did not significantly reduce the number of CD4+ or CD8+ T lymphocytes in CSF. In addition, there were no significant changes in °/} of CD4+ T lymphocytes and °/, CD8+ T lymphocytes (Table 26). Still, no significant changes in peripheral blood WBC, CD4+ and CD8+ menorial T lymphocytes (Table 27) were observed.
Table 25: Effect of treatment on CSF CD4+:CD8+ ratio (evaluable population, n=13) Low l Week 5 I [)1 difference of I CD4+:CD8+ I ratio t Ratio 3.59 (0.273) 3.60 (0.265) * 0.01 (0.197) i CD4+:CD8+ i 1.53-5.67) 1.42-5.15 | Mean range (SE) 90°/, 2-sided CI 3.00-4.19 3.132, 4.077 I for proportion joo°/, 2-sided CI I I I -0.337, 0.363 I for CI difference-confidence interval
*p<0.001 (one-sided sample t-test for H0:µ<] vs Hl: µ>=1). tDifference is defined as ratio of week 5 minus baseline ratio Table 26: Effect of treatment on CD4+ and CD8+ lymphocyte count in CSF
(evaluable population, n 1 3) Baseline Week5
CD4+ as °/, from 75.160 (7.3831) 74.215(6.3732)
lymphocytes, mean (SD) I CD8+ as "Q of I 22.272 (5.4320) I 22.007 (6.1624)
! lymphocytes, mean (SD)
Table 27: Peripheral blood [.memory T lymphocytes (RO+) counts
(Evaluable population, n=13)
Basal Sernana 5
Average (SD) Average (SD)
CD4+CD45RO+ 27.85 (4.98) 27.06 (5.02)
CD8+CD45RO+(°4) 11.24 (3.40) 10.78 (2.98)
Summary Vedolizumab did not affect CSF CD4+ and CD8+ cell counts or CD4+:CD8+ ratio in healthy volunteers after a single 450 mg dose.
None of the 10 subjects had a reduction in the CD4+:CD8+ CSF after dose ratio by less than ! Vedolizumab was not detected in CSF.
Furthermore, there was no change observed in total WBCs or CD4+ and CD8+ memory T lymphocyte subsets in peripheral blood.
Target saturation (ct4j37) in blood occurred in all subjects at the time of assessment point assessment.
The levels of CSF CD4+ and CD8+ lymphocytes and microportion were similar to those previously reported in the literal.
These results are consistent with the lack of effect of vedolizumab on both immune survival in physiological CNS and pathological CNS inflammation of monkeys (See
Example 4) Example 7: Long-term clinical experience with Vedolizumab for the treatment of IBD. An open-label, phase 2 safety extension study was completed to assess the long-term pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy of vedolizumab.
The patients were aged 18 to 75 years, and had previously participated in an early PK/PO/safety study in patients with ulcerative colitis or had symptoms of IBD for at least 2 months confirmed by endoscopy and/or histopathology and/or radiology in 36 screening cases. All patients received an intravenous dQsaf'em regimen of either 2 mg/kg or 6 mg/kg vedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125 mM sodium chloride, 0.054% polysorbate 80, pH 6.0 (long term stored -70°C and up to 3 months at -20°C)) on days 1, 15 and 43, followed by one dose every 8 weeks for up to a total of 78 weeks Patients were those with naive treatment ulcerative colitis or patients with Crohn's disease, or patients with ulcerative colitis who had participated in a previous clinical study.
The effectiveness/quality of life (QoL); Partial Mayo score (PMS), Crohn's Disease Activity Index (CDAI), and Inflammatory Bowel Disease Questionnaire (IBDQ) were used to assess the study results.
PK results Mean pre-infusion vedolizumab concentrations were dose proportional, and remained stable and detectable throughout the study. Results PD Receptors (%ACT-1 + [CD4+CD45RO HIGHJ and '/> MADCAM+ [CD4+CD45RO IJJGH] were nearly (optionally inhibited over the study period at all dose levels. Mayo score partial mean basal mean PMS) was higher for the treatment of patients with untreated ulcerative colitis (5.4) than for extended ulcerative colitis patients (2.3) By day 43, PMS mCdio showed a pronounced reduction for both patients with untreated and untreated ulcerative colitis (2.3). in extension. By day 155, mean scores of the two groups were similar. Mean PMS continued to decrease until day 267, and leveled off thereafter. Mean CDAF Crohn's Disease Activity Index of CD patients reduced from 294.6' at baseline to 237.7 on Day 43, and continued to decrease until day L55 (156.1).
IBDQ Patients with prolonged ulcerative colitis had the highest mean IBDQ scores at baseline. By day 43, mean IBDQ scores increased in all three disease groups. Average TBDQ scores continued to increase over time in all 3 disease groups, reaching a maximum on day 155 for Crohn's disease patients, and on day 491 for patients with untreated colitis and patients with extended ulcerative colitis .
C-reactive protein in both patients with ulcerative colitis and crohn's disease
5 showed mean reduced levels of CRP through day 155 and then leveled off.
Patients with untreated ulcerative colitis had a lower mean CRP level at baseline! than patients with extended ulcerative colitis (2.28 v. 7.09). Mean CRP levels of patients with untreated ulcerative colitis remained relatively constant at all evaluable time points.
Other Safety Results No systemic opportunistic infections (including PML) were reported during the study.
One patient tested positive for JC viremia at a single time point, while being negative for JCV at all other time points.
Three of 72 patients (4%) had positive HAHA results (two of these were transiently positive). The study did not show evidence of liver toxicity, lymphocytosis, or lymphopenia, or any other drug-associated disorder.
Conclusions Vedolizumab given at 2.0 or 6.0 mg/kg once every 8 weeks for up to 78 weeks reached target recipient saturations, was associated with lasting mean reductions in disease activity and improved TBDQ scores, was generally safe and well tolerated, and demonstrated acceptable immunogenicity.
Example 8: Induction and Maintenance of Response and Remission in Patients with Moderately to Severely Active Crohn's Disease A randomized, double-blind, placebo-controlled, multicenter study was completed to evaluate the induction effect of vedolizumab at doses of 300 mg (reconstituted from a formulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, 10°/q sucrose, in pH 6.3 which has been lyophilized) in patients with TNF antagonist failure week 6 (after 2 doses--O and 2 seínanas) and at week 10 (after 3 doses). The study consisted of 416 patients, 7S°/q of whom were TNFα antagonist failures, and 25°4 of whom were TNFα naive.
Demographics and concomitant IBD medication were balanced across treatment groups.
Baseline disease characteristics were further balanced across treatment groups for baseline disease, except for baseline disease activity.
The primary endpoint designed for the study was week 6 of remission (°/0) in anti-WF"-a antagonist failure population. The secondary key endpoints that were assessed (sequential testing procedure procedure) forani: week 6 remission (°/9) in the general population, week 10 remission ('/q) in anti-TNF-α 5 antagonist failure and general population (using Hochber procedure{'), week 6 and 10 remission sustained (°/9) in anti-l'NF-α antagonist failure and gerat population (using Hochberg procedure), and week 6 of improved response (°/0) in anti-TNF-α antagonist failure population.
Table 28: Basal CDAI: Vedolizumabealor acebo p I TNF ITT: Average i 306.1 (55.43) i 316.1 (52.63) I 0.0945 I (Standard deviation) Overall ITT: Average 301.3 (54.97) 313.9 (53.17). Difference Value p PLA VDZ Difference Vdor p IN·157 I /·158 I (RR) IIN·207 IN·209 I (RR) i Remission i 12.1 % I 15.2 '6 I 3.0 % I 0 .4332 I primary Sem6 III (1.2) i' rem ssd) 12.1 °6 19.1 % 6.9 % 0.0478 I secondary I ) I i (1.6) I Sem6 2' remss 12, 1 % 26.6 % 14.4 °6 0.0012 13 % 28.7 % 15.5 % <0.0001 secondary (2.2) (2.2) SemlO Remission 8.3 % 12.0 % 3 .7% 0.2755 8.2 % 15.3 % 7%(1.9) O.C249 sustained (1.4) (both Without 6&lC) Respond 22.3 °/0 39.2 °/, 16, 9°/, 0.0011 I improved ) II (1.8) (CDA 100) Table 30: Results in naive patients of Antij'NF-o antagonist (n=10l, 24%
of general) Placebo °/) Vedolizumab °/, Difference °/q 9S°/q Cl Remission week 6 !2 31.4 19.1 (3.3, 35.0) Remission week 16 35.3 19.2 ( 2.4, 35.8) 10 Table 3 1: Study results: Clinical remission at Weeks 6 and 10, Key subgroup Previous Tx failures, general ITT Subgroup Variable Placebo VDZ Difference 9S'/, Cl Any Failure N 156 155 I previous ajwk6 Remjl2.8 I 14.8 I2 I (-5.7, 9.7) I I anti-TNF (75°/, of I (°4) ITT) WkjO Rem 12.8 26.5 13.6 (4.9, 22.3) (Q/q) Failure prior to N 45 44 I immunomodulator I Wk 6 Rem I ]],1 I 31.8 I 20.7 I (-0.5, 39.7) ) I but not 1(°/,) anti-TNF (21% WklO Rem 15.6 31.8 16.3 (-1.1, 33.6) | JTT) I (°/0) Only failure N 5 9 greater than Wk6 Rem 0 33, 3 33.3 (-23.9, ! corticosteroid I (°/0) I 75.7) (3% ITT) WklO Rem 0 44.4 44.4 (-13.4, |(%) II i I 85, 3) The study showed that patients with TNF-cx antagonist failure required 3 doses 5 for induction of remission. Remission rates in patients with TNF-α antagonist failure increased between week 6 and week 10, but only for the vedolizumab (not pIacebu) group. substantially between week 6 and 10. Of the population of TNF-α antagonist failure with a high degree of disease severity, 43°4 never responded to a TNF-O antagonist, and 4°/, of loss of response.
Example 9: Induction and Maintenance of Response and Remission in Patients with Moderately to Severely Active Ulcerative Colitis A single study comprising two randomized, double-blind studies,
T_82/91 multicenter treatments designed to assess the induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis. Demographic and baseline disease characteristics were comparable across all treatment groups. 5 The induction study, using intravenous administration, compared placebo versus vedolizumab, at a dose of 300 mg reconstituted from a lyophilized formulation of 60 mg/mL antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, 1O °/, sucrose, at pH 6.3, with an endpoint at 6 weeks after 2 doses of vedolizumab.
10 The maintenance study, using the same formulation and route of administration as the induction study, compared placebo versus vedolizumab dosed every four weeks, and placebo versus vedolizumab dosed every eight weeks. Each patient was age 18-80, diagnosed with moderately to severely active ulcerative colitis, within the previous 5 year period, an inadequate response to, loss of response to, or intolerance of at least 15 conventional therapy (eg corticosteroids). s); and may receive a therapeutic dose of conventional therapies for IBD. The endpoint of this study was at 52 weeks, analyzing the population responding to the induction. Both phases of the study reached the primary endpoints, namely, clinical response in induction and clinical remission in maintenance. 20 Blood samples were collected to measure vedolizumab concentrations during the study. The serum mCdia concentration of vedolizumab at the end of the induction phase was 20 to 30 µg/mL. Mean steady-state serum vedolizimab concentrations after 30 min of lV infusion of dus:e administration of 300mg were between 9 to 13 µg/ml for the q8wks reginie and between 35 to 40 µg/ml for the reÉ' ime from q4wks. At the end of the infusion, the indian plasma concentrations of vedolizimab were between 98 and 10l µg/mL for the region of q8ks (8 weeks) and around !29 and 137 µg/mL for q4'wks (4 weeks). Summaries of response from the induction and maintenance studies are provided in Tables 32-35. A significantly greater proportion of patients treated with vedolizumab achieved clinical response, remission, and mucosal healing within 6 weeks compared to placebo (Table 32). 39% of the intention-to-treat population in the ioduction phase had previous falba anti-TNFα. Clinical response and remission rates were higher in patients on vedolizumab than on placebo among both those with prior anti4FNF failure and those without prior anti-TNF exposure. In preliminary analyzes at week 6, rates of adverse events (AES), serious AES, and adverse events leading to study discontinuation were higher in the placebo group than in the vedolizumab group.
A significantly greater proportion of patients on vedolizumab than patients on placebo achieved clinical remission, mucosal scarring, and corticosteroid-free remission within 52 weeks and response and lasting remission (Table 33). 32% of the maintenance study population had prior anti-TNFa failure. Clinical remission and durable clinical response rates were higher with vedolizumab than with placebo in both TNF and non-TNF failure patients. In the safety population CN=895) for weeks 0 to 52, the rates of adverse events (AEs), severe AES, and serious infections were similar between vedolizumab and placebo groups. No increase in the rates of opportunistic or enteric infections was observed in É'rupo vedolizumab.
Table 32: Induction study results - primary and secondary key evaluation points Placebo points Vedolizumab Ditèrence/RR PI value (I ) efficacy Response 25.5% 47.1°4 21.7%/1.8 <0.0001 i clinical è/j) Remission 5.4°4 16.9% 11.5°/o/3.1 0.0010 clinical (%) Mucosal healing 24.8% 40.9 16.1 °4/1.6 0.0013 (%) Table 33: Maintenance study results Primary and secondary key assessment points Assessment point Placebo VDZ Q8 VDZ Q4 Difference/RR Efficacy P-value N=126 N=l22 N 125 Q8 VS. Pb Q4 VS. Pb Clinical remission I 15.9 41.8 44.8 26.1/2.7 <0.0001 (O/q) 29.1/2.8 <0.0001 Durable response 23.8 56.6 52, 0 32.8/2.4 <0.0001 (%) 28.5/2.2 <0.0001 Mucosal healing (°/0) 19.8 51.6 56.0 32.0/2.6 <0.0001 i 36.3/2,& I "0.0001 Durable remission 8.7 20.5 24.0 11.8/2.4 0.0090 (°/0) III 15.3/2, 8 I 0.0011 Remission without I 13.9 I 31.4 I 45.2 17.6/2.3 I 0.0133 Corticosteriod (°/q) I n-72 I n=70 ! n 73 31.4 /3.3 l <0.000l J'abela 34: Induction study: Clinical response and remission at 6 weeks in patients with anti-TNF-a antagonist failure before and without anti-TNF exposure, population
ITT Patients with previous failure of the antij"NF-a antagonist (39%) l PIacebo point I Vedolizumab I Difference | 95°4 Cl evaluation in=63 I n=82 Response 20.6 39.0 18.4 3.9 , 32.9 clinical (%) Remission 3.2 9.8 6.6 -9.8, 22.8 clinical (Q/ú) Patients without exposure to anti-TNF-a antagonist (55°4) PIacebo Vedolizumab Difference 9S°/o Cl n=76 N=130 I Answer ! 26.3 I 53.1 I 26.8 I 13.7,39.9 ! Clinical (O/q) I Remission l 6.6 l 23.1 i 16.5 i 2.4, 30.2 i clinical (Ql) Table 35: Durable Clinical Remission and Clinical Response eni 52 Weeks: Patients with falba prior to the Anti-TNF-a antagonist or without exposure to the Anti-TNF-a antagonist in ITT population Patients with previous failure of anti-TNFmz antagonist (32°4) Assessment point Placebo VDZ VDZ Difference 95% Cl I n 38 I Q8Wks l Q4Wks I Q8wks VS II n 43 !N=40 I Placebo Q4 wks VS. Placebo Clinical remission (°/1) 5.3 37.2 35.0 31.9 10.3,
k I 29.7 51.4 7.4, 49.4 Clinical response 15.8 46.5 42.5 30.7 11.8, I durable (°/0) I | II 26.7 49.6 7.5, 45.9 Patients without exposure to the anti-TNF-a antagonist (60°4) I Piacebo I VDZ VDZ I Diterence 9S°/, Cl I n 79 I Q8Wks Q4wks I Q8wks VS .
I n=72 n=73 l Placebc I Q4wks VS.
I Placebo Clinical remission (°4) 19.0 45.8 47.9 26.8 12.4, I 29.0 41.2 14.6, 43.3 Clinical response 26.6 65.3 56.2 38.7 24.0, durable (°/,) 29.6 53.4 14.6, 44.6 Example 10: Induction and maintenance of response and remission in patients with moderately to severely active ukerative colitis A single study comprising two randomized studies, Double-blind, 5 multicenters designed to assess the induction and maintenance of response and remission in patients with moderately to severely active ulcerative colitis. Demographic and baseline characteristics of the disease were comparable across all treatment groups. The induction study, using intravenous administration, compared placebo versus 10 vcdolizumab, in a 300 ing dose reconstituted from a lyophilized formulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine, 0.06°4 polysorbate 80, 10°4 sucrose, at pH 6.3, with an assessment point at 6 weeks after 2 doses of
86/9i vedolizumab.
The maintenance study, using the same formulation and route of administration as the induction study, compared placebo versus vedolizumab dosed every four weeks, and placebo versus vedolizumab dosed every eight weeks.
The endpoint of this study was at 52 weeks, analyzing the population responding to the induction.
Surprisingly, this study showed that groups Q4 and Q8 weeks generated very similar results.
Summaries of responses from the induction and maintenance studies are provided in Tables 36 to 39. A significantly greater proportion of patients treated with vedolizumab achieved clinical remission and improved response compared to 10 with placebo (Table 36). Improved remission and response rates were greater in vedolizumab than placebo patients among both those with prior anti-jNF failure and those without prior anti-TNF exposure.
Rates of adverse events (AEs), AES E'ra'es, and serious infections were similar between the vedolizumab and placebo groups.
No increase in timely or enteric infection rates was observed in the vedolizumab group.
Table 36: Induction study results - primary and secondary evaluation points I Placebo Vedolizumab points Difference P value evaluation IN=148 N 220 adjusted /RR Remission 6.8% 14.5% 7.8%/2.1 0.0206 clinic (O/q)
Answer 25.7°4 31.4% 5.7%/1.2 0.2322 improved (°4) I
Change from -3.6 -2.9 0.9288
Mean CRP In 147 N=220 (µt'/mL)
Table 37: Maintenance study results - primary and secondary key assessment points
Placebo evaluation point VDZ Q8 VDZ Q4 Difference Efficacy value I I N=l53 I N-154 I N=!54 i adjusted IRR IP
I Q8vs.Pb
I Q4vs.Pb
' Cynic remission 21.6 39.0 36.4 17.4/1.8 0.0007
(°4) 14.7/1.7 0.0042
Improved response 30.1 43.5 45.5 13.4/1.4 0.0132
(°/9) 15.3/1.5 0.0053
Remission without 15.9 31.7 28.8 15.9/2.0 0.0154
Conicosteroid (°4) N=82 N=82 N=80 12.9/1.8 0.0450
Durable remission 14.4 21.4 16.2 7.2/1.5 0.1036
(°/9) 2.0/1.1 0.6413
Table 38: Improved clinical response and remission at 6 weeks in patients with failure of Anti-TNF-o analyst before and without exposure to AntijNF, ITT population
Patients with previous failure of anti-TNF-a antagonist (48%)
I Vedolizumab Placebo Point Difference 95% Cl
I evaluation N 70 N 105
Remission i 4.3 | 10.5 ! 6.2 (-9.1, 21.3) clinical (O/j)
Response 22.9 23.8 1.0 (-11.8, 13.7) improved (°4) I
Patients without exposure to anti-TNF-a antagonist (50%)
Placebo I Vedolizumab Difference 9S°/o Cl
) n 76 I N 130109 Remission I 17.4 I 8.2 (-1.4, 17.9)
clinic (%) Answer 30.3 42.2 11.9 (-1.9.25.8) improved (°4) I
Table 39: Clinical Remission and Improved Response at 52 Weeks: Patients with Prior Anti4NF-α Antagonist Failure or No AntÈTNF-α Antagonist Exposure in
5 ITI population" Patients with previous failure of anti-TNF-u antagonist (Si°/o)
Evaluation point Placebo VDZ VDZ Difference 95°4 Cl
N 78 Q8Wks Q4Wks Q8wks VS
N-82 n 77 Placebo Q4 wks VS.
Placebo Clinical remission 12.8 28.0 27.3 15.2 (3.0, (O/Q) 14.5 27.5) (2.0, 26.9) Answer 20.5 29.3 37, 7 8.8 (-4.6, improved(°4) 17.1 22.1) (3.1, 31.2) Patients without exposure to anti-TNF-cx antagonist (45°4) Placebo VDZ VDZ Difference 95°4 Cl I n 71 I Q8Wks l Q4wks I Q8wks VS.
I n=66 ) n 71 I Placebo I Q4wks VS.
I Placebo Clinical Remission 26.8 51.1 46.5 24.8 (8.9, (°/,) III ) 19.7 40.6) (4.2, 35.2) Answer 38.0 60, 6 53.5 22.6 (6.3, improved(%) 15.5 38.9) (-0.7, 31.7) Table 40. Sequence Summary SEQ ID NO: Sequence Shown Description 1 FIG. 1 DNA encoding humanized anti-o4B7 immunoglobulin heavy chain 2 FIG. 1 Humanized anti-α4ji lobulin heavy chain amino acid sequence 3 FIG. 2 DNA encoding humanized anti-â²4B7 immunoglobulin light chain FIG. 2 Humanized anti-a±4j37 immunoglobulin levc chain amino acid sequence FIG. 3 Mature humanized light chain of LDP-02 6 FIG. 4 Generic human kappa light chain constant ref. 7 FlG. 4 Generic murine kappa light chain constant region Referenced on page 30 ACT-I antibody heavy chain CDRI from
SYWMH mouse 9 Referenced on page 30 ACT-I antibody heavy chain CDR2 of
FTDPSESNTNYNQKFKG mouse Referenced on page 30 ACT-I antibody heavy chain CDR3
GGYDGWDYAJDY mouse ll Referenced on page 30 CDRI dc: ACT-I antibody light chain
mouse RSSQSLAKSYGNTYLS
90/9i
" 12 Referenced on page 30 Light Chain CDR2 of
GJSNRFS mouse ACT-L antibody
13 Referenced on page 30 Light Chain CDR3 of
LQGTHQPYT mouse ACT-I antibody
14 FIG. 7 human GM607 CL antibody kappa light chain variable region FIG. 7 Human CL antibody heavy chain variable region Although this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes and details can be made in this without departing from the scope of the invention encompassed by the appended claims.

权利要求:
Claims (21)
[1]
1. Stable formulation, characterized in that it comprises a mixture of a non-reducing sugar, an anti-α4β7 antibody and at least one free amino acid, wherein the formulation is in solid form and the molar ratio of non-reducing sugar to 5 antibody anti-α4β7 (mol:mol) is greater than 600:1, where the molar ratio of free amino acid to antibody is at least 250:1.
[2]
2. Formulation according to claim 1, characterized in that said formulation further comprises a buffering agent.
[3]
3. Formulation according to claim 1 or 2, characterized in that said non-reducing sugar is selected from the group consisting of mannitol, sorbitol, sucrose, trehalose and combinations thereof.
[4]
4. Formulation according to any one of claims 1 to 3, characterized in that said free amino acid is selected from the group consisting of histidine, alanine, arginine, glycine, glutamic acid and combinations thereof.
[5]
5. Formulation according to any one of claims 1 to 4, characterized in that said formulation further comprises a surfactant.
[6]
6. Formulation according to any one of claims 1 to 5, characterized in that said formulation is lyophilized and comprises at least about 5% to about 10% of anti-α4β7 antibody before lyophilization.
[7]
7. Stable formulation, characterized by the fact that it comprises a mixture of non-reducing sugar, an anti-α4β7 antibody, histidine, arginine and polysorbate 80, in which the formulation is in solid form and the molar ratio of non-reducing sugar to anti antibody -α4β7 (mol:mol) is greater than 600:1, where the molar ratio of total amino acid to antibody is at least 200:1.
[8]
8. Formulation according to any one of claims 1 to 7, characterized in that said antibody is vedolizumab.
[9]
9. Method for preparing the formulation, as defined in any one of claims 1 to 8, characterized in that it comprises keeping the product temperature below the collapse temperature during primary drying.
[10]
10. Use of the formulation, as defined in any one of claims 1 to 8, characterized in that it is in the treatment of a disease or disorder in a human patient, wherein said formulation is in an effective amount to treat the disease or disorder .
[11]
11. Use according to claim 10, characterized in that the solid formulation is dissolved in a suitable medium or solvent to make a liquid formulation before administration.
[12]
12. Use according to claim 10 or 11, characterized in that it is in the treatment of an inflammatory bowel disease.
[13]
13. Use according to claim 12, characterized in that the anti-α4β7 antibody is a humanized antibody and the formulation is administered to the patient according to the following dosage regimen: a. an initial dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion; B. followed by a second subsequent dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion about two weeks after the initial dose; ç. followed by a subsequent third dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion about six weeks after the initial dose; d. followed by a fourth and subsequent 300 mg doses of the humanized anti-α4β7 antibody as an intravenous infusion every four weeks or eight weeks after the subsequent third dose of the humanized antibody as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized anti-α4β7 antibody comprises an antigen binding region of non-human origin and at least a portion of an antibody of human origin, wherein the humanized anti-α4β7 antibody has binding specificity for the α4β7 complex, wherein the antigen binding region comprises the CDRs: Light chain: CDR1 SEQ ID NO:11 and CDR2 SEQ ID NO:12 and CDR3 SEQ ID NO:13, and Heavy chain: CDR1 SEQ ID NO:8 and CDR2 SEQ ID NO:9 and CDR3 SEQ ID NO:10.
[14]
14. Use according to claim 12 or 13, characterized by the fact that the patient has a lack of an adequate response, loss of response, or was intolerant to treatment with at least one of an immunomodulator, an antagonist of a factor alpha tumor necrosis or combinations thereof.
[15]
15. Use according to any one of claims 12 to 14, characterized in that inflammatory bowel disease is Crohn's disease or ulcerative colitis. 5
[16]
16. Use of a mixture of a non-reducing sugar, an anti-α4β7 antibody and at least one free amino acid, characterized in that it is in the preparation of a pharmaceutical formulation, as defined in any one of claims 1 to 8, for the treatment of a disease or disorder in a human patient.
[17]
17. Use according to claim 16, characterized in that the solid formulation is dissolved in a suitable medium or solvent to make a liquid formulation before administration.
[18]
18. Use according to claim 16 or 17, characterized in that it is in the treatment of an inflammatory bowel disease.
[19]
19. Use according to any one of claims 16 to 18, characterized in that the anti-α4β7 antibody is a humanized antibody and the formulation is administered to the patient according to the following dosage regimen: a. an initial dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion; B. followed by a second subsequent dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion about two weeks after the initial dose; ç. followed by a subsequent third dose of 300 mg of the humanized anti-α4β7 antibody as an intravenous infusion about six weeks after the initial dose; d. followed by a fourth and subsequent 300 mg doses of the humanized anti-α4β7 antibody as an intravenous infusion every four weeks or eight weeks after the subsequent third dose of the humanized antibody as needed; wherein the dosage regimen induces a clinical response and clinical remission in the patient's inflammatory bowel disease; and further wherein the humanized anti-α4β7 antibody comprises an antigen binding region of non-human origin and at least a portion of an antibody of human origin, wherein the humanized anti-α4β7 antibody has binding specificity for the α4β7 complex, where the antigen-binding region comprises the CDRs:
Light chain: CDR1 SEQ ID NO:11 and CDR2 SEQ ID NO:12 and CDR3 SEQ ID NO:13, and Heavy chain: CDR1 SEQ ID NO:8 and 5 CDR2 SEQ ID NO:9 and CDR3 SEQ ID NO:10.
[20]
20. Use according to any one of claims 16 to 19, characterized in that the patient has a lack of an adequate response, loss of response, or was intolerant to treatment with at least one of an immunomodulator, an antagonist of tumor necrosis factor alpha or combinations thereof.
[21]
21. Use according to any one of claims 16 to 20, characterized in that inflammatory bowel disease is Crohn's disease or ulcerative colitis.
1/10
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法律状态:
2021-05-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2021-05-25| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI N? 10196/2001, QUE MODIFICOU A LEI N? 9279/96, A CONCESS?O DA PATENTE EST? CONDICIONADA ? ANU?NCIA PR?VIA DA ANVISA. CONSIDERANDO A APROVA??O DOS TERMOS DO PARECER N? 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL N? 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVID?NCIAS CAB?VEIS. |

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2021-07-06| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

---

2021-07-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161481533P| true| 2011-05-02|2011-05-02|

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US61/481,533|2011-05-02|

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US201161550545P| true| 2011-10-24|2011-10-24|

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US61/550,545|2011-10-24|

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US201261585859P| true| 2012-01-12|2012-01-12|

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US61/585,859|2012-01-12|

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