NZ617833B2 - Formulation for anti-?4?7 antibody - Google Patents
Formulation for anti-?4?7 antibody Download PDFInfo
- Publication number
- NZ617833B2 NZ617833B2 NZ617833A NZ61783312A NZ617833B2 NZ 617833 B2 NZ617833 B2 NZ 617833B2 NZ 617833 A NZ617833 A NZ 617833A NZ 61783312 A NZ61783312 A NZ 61783312A NZ 617833 B2 NZ617833 B2 NZ 617833B2
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- antibody
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- vedolizumab
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
Disclosed is a stable pharmaceutical formulation comprising a mixture of a non-reducing sugar, an anti-?4?7 antibody and one free amino acid, wherein the formulation is lyophilised, and the molar ratio of non-reducing sugar to anti-?4?7 antibody (mole:mole) is 700:1, wherein the free amino acid to antibody molar ratio is at least 250:1, and wherein the antibody comprises a heavy chain variable region comprising a complementarity determining region (CDR1) as set forth in SEQ ID NO: 8, a CDR2 as set forth in SEQ ID NO: 9, and a CDR3 as set forth in SEQ ID NO: 10, and comprises a light chain variable region comprising a CDR1 as set forth in SEQ ID NO: 11, a CDR2 as set forth in SEQ ID NO: 12, and a CDR3 as set forth in SEQ ID NO: 13, wherein the sequences are as disclosed in the complete specification. Also disclosed is a stable pharmaceutical formulation comprising 45% to 55% sucrose (w/w), an anti-?4?7 antibody, histidine, arginine, and polysorbate 80, wherein the formulation is lyophilised, wherein the molar ratio of total amino acid to antibody is at least 400:1, and wherein the antibody comprises a light chain variable region comprising amino acids 20-131 of SEQ ID NO: 4 and a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO: 2, wherein the sequences are as disclosed in the complete specification. ntibody molar ratio is at least 250:1, and wherein the antibody comprises a heavy chain variable region comprising a complementarity determining region (CDR1) as set forth in SEQ ID NO: 8, a CDR2 as set forth in SEQ ID NO: 9, and a CDR3 as set forth in SEQ ID NO: 10, and comprises a light chain variable region comprising a CDR1 as set forth in SEQ ID NO: 11, a CDR2 as set forth in SEQ ID NO: 12, and a CDR3 as set forth in SEQ ID NO: 13, wherein the sequences are as disclosed in the complete specification. Also disclosed is a stable pharmaceutical formulation comprising 45% to 55% sucrose (w/w), an anti-?4?7 antibody, histidine, arginine, and polysorbate 80, wherein the formulation is lyophilised, wherein the molar ratio of total amino acid to antibody is at least 400:1, and wherein the antibody comprises a light chain variable region comprising amino acids 20-131 of SEQ ID NO: 4 and a heavy chain variable region comprising amino acids 20 to 140 of SEQ ID NO: 2, wherein the sequences are as disclosed in the complete specification.
Description
FORMULATION FOR ANTI-(14137 DY
RELATED APPLICATIONS
This application claims the benefit of US. ional Application
61/585,859 filed on January 12, 2012, US. Provisional Application 61/550,545 filed
on October 24, 2011, and US. Provisional Application 61/481,533 filed on May 2,
201 1. The entire contents of the foregoing applications are hereby incorporated by
reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS~Web and is hereby incorporated by reference in
its entirety. 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 e a variety of
ns for pharmaceutical applications using recombinant DNA techniques.
e ns are larger and more complex than traditional organic and inorganic
drugs (Le. possessing multiple functional groups in addition to complex three-
dimensional structures), the formulation of such proteins poses special problems.
For a protein to remain ically active, a formulation must preserve the
conformational integrity of at least a core sequence of the n's amino acids,
while at the same time protecting the protein‘s multiple functional groups from
degradation. Proteins may suffer from a lack of stability, and monoclonal and
polyclonal antibodies in particular may be relatively unstable (See e.g., Wang et (11.,
J. Pharm Sci. 96:1-26 ). A large number of formulation options are available,
and not one approach or system is suitable for all proteins. Several factors to be
considered have been ed (See e.g., Wang et a1.)
Numerous characteristics may affect a protein’s stability. In fact, even in the
case of purified antibodies, the antibody structures may be heterogeneous, which
further cates the formulation of such systems. Moreover, the excipients
included in dy formulations preferably minimize any potential immune
In the case of antibodies, preservation of the conformational integrity is even
more ant. Degradation pathways for proteins can involve chemical instability
(i.e,, any s which es modification of the protein by bond formation or
cleavage resulting in a new chemical entity) or physical instability (i.e., changes in
the higher order structure of the protein). Chemical instability is manifested in, for
example, deamidation, isomerization, hydrolysis, oxidation, fragmentation, glycan
beta elimination or disulfide exchange. al instability can result from
denaturation, aggregation, precipitation or adsorption, for example. The four most
common n degradation pathways are protein ntation, ation,
deamidation, and oxidation. Consequences of chemical or al instability of
therapeutic n include a lowering of the effective administered dose, decreased
safety of the therapy due to, for example irritation or immunological reactivity, and
more frequent manufacturing due to short shelf life.
Freeze—drying is a commonly ed technique for preserving proteins;
freeze—drying serves to remove water from the protein ation of st.
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 under
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 n formulation (Pikal M., Biopharm. 3(9)26-30 (1990) and
Arakawa et a]. Pharm. Res. 8(3):285-291 (1991)).
Several publications have disclosed generally various methods of treating
inflammatory bowel diseases, and provided dosing schemes for administration of
agents designed to treat inflammatory bowel disease. For example, WO 96/24673
discloses mucosal vascular addressins and treatment of diseases ated with
leukocyte recruitment to the gastrointestinal tract as a result of leukocyte binding to
cells expressing MAdCAM. US. 2005/0095238 describes methods of treating a
disease associated with leukocyte infiltration of mucosal tissue and administration to
a human an effective amount of a human or humanized immunoglobulin or antigen
binding fragment having binding specificity for (1467 integrin. US. 2005/0095238
DM~US 33489877-6 079259 0615
further bes various doses (e.g. 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mg
immunoglobulin or fragment per kg body weight) and various intervals between doses (7,
14, 21, 28, or 30 days). However, the aforementioned patents and publications do not
disclose specific formulations of the anti-α4β7 antibody or the specific doses and dose
regimens described and claimed herein. Importantly, the aforementioned patents do not
disclose ations, doses, and dose regimens that provide for the methods of treatment
(supported by clinical trial data) described and claimed herein.
The antibody formulations of the present invention may be useful for inhibiting
leukocyte binding to cells expressing MAdCAM and therefore aid in treatment of
inflammatory bowel diseases in patients. There is, accordingly, an urgent need to discover
suitable dosages and dosing schedules of these nds, and to develop formulations,
preferably intravenous ations, which give rise to steady, therapeutically effective
blood levels of the antibody formulations over an extended period of time in a stable and
convenient form.
SUMMARY OF THE INVENTION
The invention relates to the identification of a non-reducing sugar and at least one
amino acid, as useful excipients for formulating anti-α4β7 antibody formulations whose
instability makes them susceptible to deamidation, oxidation, isomerization and/or
aggregation. The formulation improves stability, reduces aggregate ion and retards
degradation of the antibody therein.
Thus, in one aspect, the invention relates to a stable formulation comprising a
mixture of a non-reducing sugar, an 4β7 antibody and at least one free amino acid,
and the molar ratio of non-reducing sugar to anti-α4β7 antibody (mole:mole) is greater
than 600:1. The formulation may be a liquid ation or a dry formulation (e.g.,
lyophilized). The formulation can also n a ing agent. In some ments,
the non-reducing sugar is ol, sorbitol, sucrose, trehalose, or any combination
In a further aspect, the invention relates to a stable pharmaceutical formulation
comprising a mixture of a non-reducing sugar, an anti-α4β7 antibody and one free amino acid,
wherein the formulation is lyophilized, and the molar ratio of non-reducing sugar to anti-α4β7
antibody (mole:mole) is 700:1, wherein the free amino acid to antibody molar ratio is at least
- 3a -
250:1, and wherein the dy comprises a heavy chain variable region comprising a
complementarity determining region (CDR1) as set forth in SEQ ID NO:8, a CDR2 as set
forth in SEQ ID NO:9, and a CDR3 as set forth in SEQ ID NO:10, and comprises a light chain
variable region comprising a CDR1 as set forth in SEQ ID NO:11, a CDR2 as set forth in SEQ
ID NO:12, and a CDR3 as set forth in SEQ ID NO:13.
In some embodiments, the free amino acid of the formulation is histidine, alanine,
arginine, glycine, glutamic acid, or any combination thereof. The ation can comprise
between about 50 mM to about 175 mM of free amino
acid. The formulation can se between about 100 mM and about 175 mM of
free amino acid. The ratio of free amino acid to antibody molar ratio can be at least
250:1.
The ation can also contain a tant. The surfactant can be
polysorbate 20, rbate 80, a poloxamer, or any combination thereof.
In some s, the formulation can minimize immunogenicity of the anti—
(14137 antibody.
The formulation, e. g., in the dried state, can be stable for at least three
months at 40 0C, 75% relative humidity (RH).
In another aspect, the formulation is lized and comprises at least about
% to about 10% anti-(14137 antibody before lyophilization. The formulation
contain at least about 6% anti-0L4B7 antibody before lyophilization. The formulation
can be reconstituted from a lyophilized formulation (e.g., reconstituted to se
stable liquid formulation).
In another aspect, the invention relates to a stable formulation comprising
mixture of a non—reducing sugar, an anti-0L4B7 antibody and at least one free amino
acid, and the molar ratio of non-reducing sugar to 14137 antibody (molezmole) is
greater than 600:1 and the ratio of free amino acid to anti-(14137 antibody
(mole2mole) 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-(14137 antibody
and at least one free amino acid, wherein the molar ratio of non-reducing
sugar to
anti-(14137 antibody (molezmole) is greater than 600: 1. In yet a further aspect, the
invention concerns a liquid formulation comprising at least about 40 mg/ml to about
80 mg/ml L4B7 antibody, at least about 50-175 mM of one or more amino acids,
and at least about 6% to at least about 10% (w/v)
sugar. The liquid formulation may
also contain a buffering agent. In some embodiments the liquid formulation also
comprises a metal chelator. In some embodiments, the liquid formulation also
comprises an anti-oxidant.
In another aspect, the invention relates to a liquid formulation comprising at
least about 60 mg/ml anti-(14137 antibody, at least about 10% (w/v) non-reducing
sugar, and at least about 125 mM of one or more free amino acids.
DM_US 33489871607959 0615
In another aspect, the invention relates to a liquid formulation comprising at least
about 60 mg/ml anti-α4β7 dy, at least about 10% (w/v) non-reducing sugar, and at
least about 175 mM of one or more free amino acids
In still yet a further aspect, the invention also relates to a dry, e.g., lyophilized
formulation comprising a mixture of a ducing sugar, an anti-α4β7 dy,
histidine, arginine, and polysorbate 80, wherein the formulation is in solid form, and the
molar ratio of non-reducing sugar to anti-α4β7 antibody (mole:mole) is greater than 600:1.
In still yet a further aspect, the invention relates to a lized ation
comprising a mixture of a non-reducing sugar, an anti-α4β7 antibody, ine, arginine,
and rbate 80. In this aspect, the molar ratio of non-reducing sugar to anti-α4β7
antibody mole) is greater than 600:1. Furthermore, the molar ratio of arginine to
4β7 antibody (mole:mole) in the formulation is greater than 250:1.
In another aspect, the invention relates to a method of making a pharmaceutical
formulation described herein, comprising mixing a non-reducing sugar, an anti-α4β7
antibody and arginine, lyophilizing the mixture, and maintaining the product temperature
below the collapse temperature during primary drying. The method can also contain an
annealing step.
In one aspect, the invention relates to a method for treating a human patient suffering
from inflammatory bowel disease, wherein the method comprises the step of administering to
a patient suffering from inflammatory bowel disease, a humanized immunoglobulin or
antigen-binding fragment thereof having binding icity for human α4β7 in, wherein
the zed immunoglobulin or n-binding fragment comprises an antigen-binding
region of nonhuman origin and at least a portion of an antibody of human origin, wherein the
humanized 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 humanized
immunoglobulin or antigen-binding fragment thereof as an intravenous infusion; (b) followed
by a second subsequent dose of 300 mg of the humanized immunoglobulin or antigen-binding
fragment thereof as an intravenous infusion at about two weeks after the initial dose; (c)
followed by a third subsequent dose of 300 mg of the humanized immunoglobulin or antigen-
g fragment thereof as an
intravenous infusion at about six weeks after the initial dose; (d) followed by a
fourth and subsequent doses of 300 mg of the humanized immunoglobulin or
antigen-binding fragment f as an intravenous infusion every four weeks or
every eight weeks after the third subsequent dose of the humanized antibody as
needed; wherein the dosing regimen induces a clinical response and/or clinical
remission in the atory bowel disease of the patient; and further wherein the
zed immunoglobulin or antigen-binding nt has g specificity for
the a4B7 complex, wherein the antigen-binding region comprises three
complementarity determining regions (CDRI, CDR2, and CDR3) of a light chain
variable region and three complementarity determining regions (CDRI, CDR2, and
CDR3) of a heavy chain variable region of the amino acid sequence set forth below:
light chain: CDRl SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ ID NO:11;
heavy chain: CDRl SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3 SEQ ID NO:14.
In another aspect, the invention relates to a dosing regimen for the
therapeutic treatment of inflammatory bowel disease, wherein the dosing regimen
comprises the step of: administering to a patient suffering from inflammatory bowel
disease, a humanized immunoglobulin or antigen—binding fragment thereof having
binding city for human (x4137 integrin, wherein the humanized
globulin or antigen-binding fragment comprises an antigen-binding region
of nonhuman origin and at least a portion of an antibody of human origin, n
the humanized immunoglobulin or antigen-binding fragment thereof is stered
to the patient according to the following dosing n: (a) an initial dose of 300
mg of the humanized immunoglobulin or n-binding fragment thereof as an
intravenous infusion; (b) followed by a second subsequent dose of 300
mg of the
humanized immunoglobulin or n~binding fragment thereof as an intravenous
infusion at about two weeks after the initial dose; (c) followed by a third subsequent
dose of 300 mg of the humanized immunoglobulin or n-binding fragment
thereof as an intravenous infusion at about six weeks after the initial dose; (d)
followed by a fourth and subsequent doses of 300 mg of the humanized
immunoglobulin or antigen-binding fragment f as an intravenous infusion
every four weeks or every eight weeks after the third subsequent dose of the
humanized antibody as needed; wherein the dosing regimen induces a clinical
DMiUS 33489877-6 079259 0615
response and/or clinical remission in the inflammatory bowel disease of the patient;
and r wherein the zed immunoglobulin or antigen-binding fragment has
binding specificity for the a4B7 complex, wherein the antigen-binding region
comprises three complementarity determining regions (CDRl , CDRZ, and CDR3) of
a light chain variable region and three complementarity determining s (CDRl,
CDRZ, and CDR3) of a heavy chain variable region of the amino acid sequence set
forth below: light chain: CDRl SEQ ID NO:9, CDRZ SEQ ID NO: 10, CDR3 SEQ
ID NO:11; heavy chain: CDRl SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3 SEQ
ID NO: 14.
In some s the method of ent with the anti—a4B7 antibody
formulation, the dose, or the dose regimen can minimize immunogenicity of the
anti-a4B7 antibody.
The patient may have had a lack of an adequate
se with, loss response
to, or was intolerant to treatment with at least one of an modulator, a tumor
necrosis factor-alpha (TNF-a) antagonist or combinations thereof.
The inflammatory bowel disease can be Crohn’s disease or ulcerative colitis.
The inflammatory bowel disease can be moderate to severely active ulcerative
colitis.
The dosing regimen can result in mucosal healing in patients suffering from
moderate to severely active ulcerative colitis.
The patient may have usly received treatment with at least one
corticosteroid for the inflammatory bowel disease. The dosing regimen can result in
a reduction, elimination or reduction and elimination of corticosteroid use by the
patient.
In some s, the humanized immunoglobulin or antigen-binding
nt thereof is administered in a final dosage form at a concentration of
between about 1.0 mg/ml to about 1.4 mg/ml. The humanized immunoglobulin or
antigen—binding fragment thereof can be administered in a final dosage form of
about 1.2 mg/ml. The humanized immunoglobulin or antigen—binding fragment can
be administered to the patient in about 30 minutes.
The humanized immunoglobulin or antigen-binding fragment thereof can be
reconstituted from a lyophilized formulation.
DMiUS 33489877-6 0792590615
The humanized immunoglobulin or antigen-binding fragment thereof can be
reconstituted to comprise a stable liquid formulation.
In some aspects, the dosing regimen does not alter the ratio of CD4 to CD8
in cerebrospinal fluid of patients receiving said treatment.
The patient can be a person 65 years of age or older and does not require
adjustment of the dosing n.
BRIEF DESCRIPTION OF THE DRAWINGS
is an illustration of a tide sequence (SEQ ID NO: 1) encoding
the heavy chain of a humanized anti-a4B7 immunoglobulin, and the deduced amino
acid ce of the heavy chain (SEQ ID N0:2). The tide
sequence contains
cloning sites (lower case), Kozak sequence (upper case, nucleotides 18-23 of SEQ
ID NO: 1) and leader sequence (lower case, nucleotides 24-86 of SEQ ID NO: 1) at
the 5' end of the heavy chain. The open reading frame of the nucleotide
sequence is
nucleotides 24-1433 of SEQ ID N0zl.
is an illustration of a nucleotide sequence (SEQ ID N023) encoding
the light chain of a humanized immunoglobulin referred to herein as vedolizumab,
and the deduced amino acid sequence (SEQ ID N0: 4) of the light chain. The
nucleotide sequence contains cloning sites (lower case), Kozak
ce (upper
case, nucleotides 18-23 of SEQ ID N03) and leader sequence (lower case,
nucleotides 24-80 of SEQ ID N023) at the 5' end of the heavy chain. The
open
reading frame of the nucleotide sequence is nucleotides 24—737 of SEQ ID N023.
is an alignment of the amino acid sequences of (A) the mature
humanized light chain (amino acids 20-238 of SEQ ID N0:4) of the humanized
immunoglobulin ed to herein as zumab and (B) the mature humanized
light chain of the humanized immunoglobulin referred to herein as LDP—02 (SEQ ID
N0:5). (Regarding LDP-02, see, W0 98/06248 and Feagan et al., N. Eng. J. Med.
352:2499-2507 (2005). Feagan et al. describe a clinical study of , but in the
e they refer to LDP—02 as MLN02.) The alignment illustrates that the amino
acid sequences of the light chains of vedolizumab and LDP-02 differ at positions
1 l4 and l 15 ofthe mature light chains.
DM‘US 33489877-6 0792590015
is an alignment of amino acid sequences of (A) a generic human
kappa light chain constant region (SEQ ID NO:6) and (B) a generic murine kappa
light chain constant region (SEQ ID NO:7). The amino acid residues Thr and Val
(which are present at ons 114 and 115 of the mature vedolizumab light chain
(amino acids 133 and 134 of SEQ ID NO:4)) are present in the constant region of
the human kappa light chain, whereas the amino acid residues Ala and Asp (which
are present at ons 1 14 and 1 15 of the mature LDP-OZ light chain (SEQ ID
NO:5)) are present in the constant region of the mouse kappa light chain.
is a map of vector pLKTOK38D (also referred to as pTOK38MLN02-
TV), which encodes the humanized heavy chain and the humanized light chain of
MLNOZ, and is suitable for producing vedolizumab in CHO cells. (See, US. Patent
Application Publication No. 2004/0033561 A1 which ses pLKTOK3 8.
pLKTOK38D is a variant OK38 in which the restriction sites indicated on
the map flank the sequence encoding the light Chain variable region.)
shows the predicted models for change in percent
monomer, change
in percent aggregate, and change in percent major isoform of the anti—(1437
lized formulation. The models are based on statistical analysis of the data
ted in Example 1. The center line shows the results for the predictive models
and the outer lines show the 95% confidence limit for the predictive models. 6B shows alternative models based on the statistical analysis of 40°C data from
Tables 1—3 when the input factors are pH, sugarzprotein molar ratio, and
nezprotein molar ratio. The center line shows the results for the predictive
models and the outer lines show the 95% confidence limit for the predictive models.
shows the amino acid sequences of (A) the mature human GM607’CL
dy kappa light chain variable region and (B) the human 21/28’CL heavy chain
variable region.
is a graph showing that solids and load affect drying time (the
numbers in the lines represent the number of minutes of drying time).
is a graph showing vedolizumab did not did not delay onset of clinical
symptoms of experimental autoimmune encephalomyelitis (EAE) as compared to
placebo l. Natalizumab significantly (p<0.05) delayed onset of clinical
symptoms of EAE as compared to placebo control.
DMkUS 77-6 0792590615
ED DESCRIPTION OF THE INVENTION
The invention relates to formulations comprising anti-0L4B7 antibodies. The
formulations may be mixtures comprising non-reducing
sugar, anti-a4B7 antibody
and one or more free amino acids, and the molar ratio of the non-reducing
sugar to
anti-a4B7 antibody is greater than 600 mole non—reducing sugarzl mole anti-(14137
antibody. The formulations may be in a solid or liquid form.
Definitions
The term “pharmaceutical formulation” refers to a preparation that contains
an 14137 antibody in such form as to permit the biological ty of the
antibody to be effective, and which contains no additional ents which are
unacceptably toxic to a subject to which the formulation would be administered.
A “stable” formulation is one in which the antibody n substantially
retains its physical stability and/or al stability and/or its biological activity
upon storage. In one aspect, the formulation substantially retains its physical and
chemical stability, as well as its biological activity
upon storage. The storage period
is generally selected based on the intended life of the formulation. Various
analytical techniques for measuring protein ity are available in the art and are
reviewed in Peptide and Protein Drug Delivery, 247—301, Vincent Lee Ed., Marcel
, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev.
: 29-90 (1993), for example.
A “deamidated” monoclonal antibody is one in which one or
more
asparagine or glutamine residue thereof has been derivatized, e. g. to an aspartic acid
or an iso-aspartic acid.
An antibody which is ptible to ation” is one comprising
one or
more residue which has been found to be prone to deamidate.
An antibody which is “susceptible to oxidation” is an antibody comprising
one or more residue which has been found to be prone to oxidation.
An antibody which is “susceptible to aggregation” is one which has been
found to aggregate with other dy molecule(s), especially
upon freezing,
heating, drying, reconstituting and/or agitation.
DMfiUS 33489877-6 0792590615
An antibody which is ptible to fragmentation” is one which has been
found to be cleaved into two or more fragments, for example at a hinge region
thereof.
By “reducing deamidation, oxidation, aggregation, or fragmentation” is
intended to mean preventing or decreasing (e.g., to 80%, 60%, 50%, 40%, 30%,
% or 10% of) the amount of deamidation, aggregation, or fragmentation relative
to the monoclonal antibody formulated at a ent pH or in a different buffer.
An “aggregate”, “SEC aggregate”, or “soluble ate” is more than one
and less than or equal to ten dy proteins and/or fragments associated together
through either covalent, ionic, or hydrophobic interactions to form a larger protein
body.
An “insoluble aggregate” or “particle” is greater than ten antibody proteins
and/or fragments associated together through either covalent, ionic, or hydrophobic
interactions to form a larger protein body.
As used herein, “biological ty” of a monoclonal antibody refers to the
ability of the antibody to bind to antigen and result in a measurable biological
response which can be measured in vitro or in vivo. Such activity may be
antagonistic or agonistic.
The cell surface molecule, “0L4B7 integrin,” or “a467,” is a heterodimer of
an a4 chain (CD49D, ITGA4) and a [37 chain ). Each chain can form a
heterodimer with an alternative in chain, to form a4l31 or OLEB7. Human
0L4 and
[37 genes (GenBank (National Center for Biotechnology ation, Bethesda, MD)
RefSeq Accession numbers NM“000885 and NM_000889, tively) are
expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes.
Typical of many integrins, (x4B7 can exist in either a resting or ted state.
Ligands for 0L4B7 include vascular cell adhesion molecule (VCAM), fibronectin and
mucosal addressin (MAdCAM (e. g., MAdCAM—1)).
As used herein, a human immunoglobulin or antigen-binding nt
thereof that has “binding specificity for the a4B7 complex” binds to (14W, but not to
0L4Bl or aEB7.
DMiUS 33489877~007925906|5
As used , an “isotonic” formulation has substantially the same osmotic
re as human blood. Isotonic formulations will generally have an osmotic
pressure from about 250 to 350 mOsm. Isotonicity can be ed using a vapor
pressure or eezing type osmometer, for example.
As used herein, ring agent” refers to a buffer that resists changes in pH
by the action of its acid-base conjugate components. The buffering agent may be
present in a liquid or solid formulation of the invention. The buffering 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 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, ate, e, cacodylate, 2—[N-
morpholino]ethanesulfonic acid (MES), bis(2—
hydroxyethyl)iminotris[hydroxymethyl]methane (Bis—Tris), N—[2-acetamido]
iminodiacetic acid (ADA), glycylglycine and other organic acid buffers. In another
I5 aspect, the buffering agent herein is histidine or citrate.
A “histidine buffer” is a buffer comprising histidine ions. Examples of
histidine buffers include histidine chloride, histidine acetate, histidine phosphate,
histidine sulfate solutions. The histidine buffer or histidine-HCl buffer has a pH
between about pH 5.5 to 6.5, about pH 6.1 to 6.5, or about pH 6.3.
A “saccharide” herein is a compound that has a general formula n and
derivatives thereof, including monosaccharides, disaccharides, trisaccharides,
polysaccharides, sugar alcohols, reducing sugars, nonreducing , and the like.
In one aspect, examples of saccharides herein include glucose,
sucrose, trehalose,
lactose, fructose, maltose, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol,
mannitol, mellibiose, tose, raffinose, mannotriose, stachyose, maltose,
lactulose, maltulose, glucitol, ol, lactitol, iso-maltulose, and the like. A
saccharide can be a lyoprotectant. In another aspect, the saccharide herein is a
nonreducing disaccharide, such as sucrose.
A ctant” herein refers to an agent that lowers surface tension of a
liquid. The surfactant can be a nonionic surfactant. In one aspect, examples of
surfactants herein e polysorbate (polyoxyethylene sorbitan monolaurate, for
e, polysorbate 20 and, polysorbate 80); TRITON (t—
DM_US 33489877-607925906I 5
Octylphenoxypolyethoxyethanol, nonionic detergent, Union Carbide iary of
Dow Chemical Co., Midland MI); sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl—, linoleyl-, or stearyl-sulfobetaine;
lauryl-, myristyl-, yl- or l-sarcosine; linoleyl-, myristyl—, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl—,
opropylg or isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine;
sodium methyl c0coyl-, or disodium methyl oleyl-taurate; sorbitan monopalmitate;
and the MONAQUAT series (Mona Industries, Inc., Paterson, NJ); polyethyl
glycol (PEG), polypropylene glycol (PPG), and copolymers of poloxyethylene and
poloxypropylene glycol (e.g. Pluronics/Poloxamer, PF68 etc); etc. In another
aspect, the surfactant is polysorbate 80.
The term “antibody” herein is used in the broadest sense and specifically
covers full length monoclonal antibodies, globulins, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from at least two full
length antibodies, e. g., each to a ent antigen or epitope, and individual n
binding fragments, including dAbs, scFv, Fab, F(ab)'2, Fab', including human,
humanized and dies from non—human species and recombinant antigen binding
forms such as monobodies and diabodies.
Molar amounts and ratios of anti-(1407 antibody to other excipients described
herein are ated on the assumption of an approximate molecular weight of
about 150,000 daltons for the antibody. The actual antibody molecular weight may
differ from 150,000 daltons, depending on amino acid composition or post—
translational modification, e.g., as ent on the cell line used to
express the
antibody. Actual antibody molecular weight can be +/- 5% of 150,000 daltons.
The term “human antibody” includes an antibody that
possesses a sequence
that is derived from a human germ—line immunoglobulin
ce, such as an
antibody derived from enic mice having human immunoglobulin genes (e.g.,
XENOMOUSE genetically engineered mice (Abgenix, Fremont, CA), HUMAB—
MOUSE ®, KIRIN TC M transchromosome mice, KMMOUSE®
(MEDAREX, Princeton, NJ)), human phage display libraries, human myeloma cells,
or human B cells.
DM_US 33489877-6 079259 0615
The term “monoclonal antibody” as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e., the
dual antibodies comprising the population are identical and/or bind the same
epitope, except for possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor amounts. In
contrast to polyclonal antibody preparations that lly include different
antibodies directed t ent determinants (epitopes), each monoclonal
antibody is ed against a single determinant on the antigen. The modifier
“monoclonal” indicates the character of the antibody as being obtained from a
ntially homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For example, the
monoclonal dies to be used in accordance with the present invention
may be
made by the hybridoma method first described by Kohler et al., , 256:495
(1975), or may be made by recombinant DNA s (see, e. g., US. Pat. No.
4,816,567). The “monoclonal antibodies” may also be ed from phage antibody
libraries using the techniques described in Clackson et al., Nature, 3522624-628
(1991) and Marks et al., J. M01. Biol, 222:581-597 (1991), for e.
The 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
belonging to a particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to ponding sequences in antibodies
derived from another s or belonging to another antibody class or subclass, as
well as nts of such antibodies, so long as they exhibit the desired biological
activity (U.S. Pat No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984)). 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.
“Antigen binding fragments” of the humanized immunoglobulin prepared in
the formulation of the invention comprise at least the le regions of the heavy
and/or light chains of an anti—a4B7 antibody. For example, an antigen binding
DM_US 33489877-6 07925906] 5
fragment of vedolizumab comprises amino acid residues 20—131 of the humanized
light chain sequence of SEQ ID N024. es of such antigen binding fragments
include Fab fragments, Fab' fragments, scFv and F(ab')2 fragments of a humanized
immunoglobulin known in the art. Antigen binding fragments of the humanized
immunoglobulin of the invention can be produced by enzymatic cleavage or by
recombinant techniques. For instance, papain or pepsin ge can be used to
generate Fab or F(ab')2 fragments, respectively. Antibodies can also be produced in
a variety of truncated forms using antibody genes in which one or more stop codons
have been introduced am of the natural stop site. For example, a recombinant
construct encoding the heavy chain of an F(ab')2 fragment can be designed to
include DNA sequences ng the CH1 domain and hinge region of the heavy
chain. In one aspect, antigen binding fragments inhibit binding of a4B7 in to
one or more of its ligands (e. g. the mucosal addressin MAdCAM (e.g.,MAdCAM—
l), fibronectin).
Papain digestion of antibodies produces two identical antigen binding
fragments, called “Fab” fragments, each with a single antigen binding site, and a
residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin
treatment yields an F(ab')2 fragment that has two antigen binding sites and is still
capable of cross-linking antigen.
“Fv” is an antibody fragment which consists of a dimer of one heavy chain
variable domain and one light chain variable domain in non-covalent association.
The Fab fragment also contains the constant domain of the light chain and
the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy us of the heavy
chain CH1 domain including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine e(s) of the
constant domains bear at least one free thiol group. F(ab')2 antibody nts
originally were produced as pairs of Fab' nts which have hinge nes
between them. Other chemical couplings of antibody fragments are also known.
“Single-chain Fv” or “scFv” dy fragments comprise the VH and VL
domains of antibody, n these domains are present in a single polypeptide
chain. In one , the Fv polypeptide further ses a polypeptide linker
DM_US 33489877—6079259061 5
between the VH and VL domains which enables the scFv to form the desired
structure for antigen binding. For a review of scFv see Pluckthun in The
Pharmacology clonal Antibodies, vol. 113, urg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
The term “diabodies” refers to small antibody fragments with two antigen
binding sites, which fragments comprise a variable heavy domain (VH) connected to
a le light domain (VL) in the same ptide chain (VH-VL). By using a
linker that is too short 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 described more fully in, for example,
B? 404,097; W0 93/ 1 1 161; and Hollinger er al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
A “full length antibody” is one which comprises an antigen g variable
region as well as a light chain constant domain (CL) and heavy chain constant
s, CH1, Cm and CH3. The constant domains may be native sequence constant
s (e. g. human native sequence constant domains) or amino acid
sequence
ts thereof. In one aspect, the full length antibody has one or more effector
functions.
An “amino acid sequence variant” antibody herein is an antibody with an
amino acid sequence which differs from a main species antibody. Ordinarily, amino
acid sequence variants will s at least about 70%, at least about 80%, at least
about 85%, at least about 90%, or at least about 95% homology with the main
species antibody. The amino acid sequence variants possess substitutions, ons,
and/or additions at n positions within or adjacent to the amino acid
sequence of
the main species antibody, but retain n binding activity. Variations in
sequence of the constant regions of the antibody will have less effect on the antigen
binding activity than variations in the variable regions. In the variable regions,
amino acid sequence variants will be at least about 90% homologous, at least about
95% homologous, at least about 97% homologous, at least about 98% homologous,
or at least about 99% homologous with the main species antibody.
“Homology” is defined as the percentage of residues in the amino acid
sequence variant that are identical after aligning the sequences and introducing gaps,
DM_US 33489877-6 079259.06I 5
if necessary, to achieve the maximum percent homology. Methods and er
programs for the 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-
a4l37 antibodies.
A “glycosylation variant” antibody herein is an antibody with one or more
carbohydrate moeities attached thereto which differ from one or more carbohydrate
moieties ed to a main species antibody. Examples of glycosylation ts
herein include antibody with a G1 or G2 oligosaccharide structure, instead of a G0
oligosaccharide structure, attached to an Fc region thereof, antibody with one or two
carbohydrate moieties attached to one or two light chains thereof, antibody with no
carbohydrate attached to one or two heavy chains of the antibody, etc, and
combinations of glycosylation alterations.
Antibody “effector functions” refer to those biological ties attributable
to the Fc region (a native sequence Fc region or amino acid sequence t Fc
region) of an antibody. Examples of antibody effector functions include C lq
binding; complement dependent cytotoxicity; Fc receptor binding; antibody-
dependent cell—mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g. B cell receptor; BCR), and the like.
Depending on the amino acid ce of the constant domain of their heavy
chains, full length antibodies can be ed to ent “classes”. There are five
major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3,
IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the
different s of antibodies are called or, 6, a, y, and p, respectively. The subunit
structures and three—dimensional configurations of different classes of
globulins are well known.
The “light chains” of antibodies from any vertebrate species can be assigned
to one of two y distinct types, called kappa (K) and lambda (it), based on the
amino acid sequences of their nt domains.
“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a
cell-mediated on in which nonspecific cytotoxic cells that express Fc ors
DMAUS 33489877’6_079259.06|5
(FcRs) (e. g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas
monocytes s FcyRI, FcyRII and I. FCR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol 9:457—92 (1991). To assess ADCC activity of a molecule of interest, an in
vitro ADCC assay, such as that described in US. Pat. Nos. 5,500,362 or 5,821,337
may be performed. Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and l Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be assessed in viva, e.g.,
in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1998).
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 sequence human
FcR. In another aspect, the FcR is one which binds an IgG antibody (a gamma
receptor) and includes ors of the FcyRI, FcyRII, and FcyRIII subclasses,
including allelic ts and alternatively spliced forms of these receptors. FcyRII
receptors include A (an “activating receptor”) and B (an “inhibiting
receptor”), which have similar amino acid sequences that differ primarily in the
cytoplasmic domains thereof. Activating receptor FcyRIIA contains an
immunoreceptor tyrosine-based activation motif (ITAM) in its asmic domain.
Inhibiting receptor FcyRIIB contains an receptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (See review in M. , Annu. Rev.
Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.
l 9:457—92 ; Capel er al., Immunomethods 4:25-34 (1994); and de
Haas et al., J Lab. Clin. Med. 126:33—41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term “FCR” herein. The term also
includes the neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,
J. Immunol. 24:249 (1994)).
The term “hypervariable region” when used herein refers to the amino acid
residues of an antibody which are responsible for antigen g. The
DMiUS 33489877-60792590615
ariable region generally comprises amino acid residues from a
“complementarity ining region” or “CDR” (e.g. residues 24—34 (L1), 50~56
(L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)
and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins ofImmunological st, 5th Ed. Public Health e, al
Institutes of Health, Bethesda, Md. ) and/or those residues from a
“hypervariable loop” (eg. residues 26-32 (Ll), 50—52 (L2) and 91—96 (L3) in the
light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the
heavy chain variable domain; Chothia and Lesk J. M01. Biol. 1961901917 (1987)).
“Framework ” or “FR” residues are those variable domain residues other than
the hypervariable region residues as herein defined. The hypervariable region or the
CDRs thereof can be transferred from one antibody chain to another or to another
protein to confer antigen binding specificity to the resulting (composite) antibody or
binding protein.
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric
antibodies that contain minimal ce derived from non~human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins (recipient
antibody) in which residues from a hypervariable region of the ent are replaced
by residues from a hypervariable region of a non-human species (donor antibody)
such as mouse, rat, rabbit or nonhuman e having the desired specificity,
y, and capacity. In some instances, framework region (FR) residues of the
human immunoglobulin are replaced by corresponding non—human residues.
Furthermore, humanized antibodies may comprise residues that 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 will comprise
substantially all of at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond 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 also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that of a
human globulin. For further details, see Jones et 61]., Nature 321 :522-525
DMAUS 33489877-6 079259 0m 5
(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. 0p. Struct.
Biol. 2:593-596 (1992).
An “affinity matured” antibody is one with one or more alterations in one or
more hypervariable regions thereof which result an improvement in the affinity of
the dy for antigen, compared to a parent antibody which does not possess
those alteration(s). In one aspect, y matured antibodies will have lar
or even picomolar affinities for the target antigen. y matured antibodies are
produced by procedures known in the art. Marks et a1. Bio/Technolog/ 10:779-783
(1992) describes affinity tion by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework es is described by: Barbas et al. Proc
Nat. Acad. Sci, USA 91 :3809—3813 (1994); Schier et a]. Gene 169:147-155 (1995);
Yelton et al. J. Immunol. 155:1994-2004 (1995); n et al., J, Immunol.
154(7):3310-9 (1995); and Hawkins et al., J. M01. Biol. 226:889-896 (1992).
An “isolated” antibody is one which has been identified and ted 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, more than 99% by weight, (2)
to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino
acid sequence by use of a spinning cup sequenator, or (3) to neity by SDS-
PAGE under reducing or nonreducing conditions using Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within recombinant cells since
at least one component of the antibody's natural environment will not be present.
Ordinarily, however, ed dy will be prepared by at least one purification
step.
ment” refers to both therapeutic treatment and prophylactic or
preventative measures. Those in need of ent include those y with the
disease as well as those in which the disease or its recurrence is to be prevented.
Hence, the patient to be treated herein 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 which is formulated is substantially pure and desirably
substantially homogeneous (i.e. free from contaminating proteins etc).
DMgUS 3348987745 0792590615
“Substantially pure” antibody means a ition comprising at least about 90%
antibody by weight, based on total weight of the protein in the composition, at least
about 95% or 97% by weight. “Substantially homogeneous” antibody means a
composition sing protein wherein at least about 99% by weight of protein is
specific antibody, e.g., anti-a4fi7 dy, based on total weight of the protein.
“Clinical remission” as used herein with reference to ulcerative colitis
subjects refers to a te Mayo score of 2 or less points and no individual
subscore greater than 1 point. Crohn’s disease “clinical remission” refers to a CDAI
score of 150 points or less.
A “clinical response” as used herein with reference to ulcerative colitis
subjects refers to a ion in complete Mayo score of 3 or greater points and 30%
from baseline, (or a partial Mayo score of 2 or greater points and 25% or greater
from baseline, if the complete Mayo score was not performed at the visit) with an
accompanying decrease in rectal bleeding subscore of 1 or greater points or absolute
rectal bleeding score of 1 or less point. A “clinical response” as used herein with
reference to s disease subjects refers to a 70 point or greater decrease in
CDAI score from baseline (week 0).
“Mucosal g” as used herein with reference to ulcerative colitis subjects
refers to an endoscopic subscore of 1 point or less.
As used herein, “treatment failure” refers to disease worsening, a need for
rescue medications or surgical intervention for treatment of ulcerative colitis or
Crohn’s disease. A rescue medication is any new medication or any increase in dose
of a baseline tion required to treat new or lved tive colitis or
Crohn’s disease symptoms (other than antidiarrheals for control of c ea).
Formulations
As described herein, it has been discovered that anti-04W antibodies are
highly stable when in a dry, e. g, lyophilized formulation with excess (on a mole
basis) non-reducing sugar. In particular, lyophilized formulations in which the ratio
of non-reducing sugar to anti- d4B7 antibody (molezmole) is greater than 600:1 are
shown herein to be stable for at least 2 years.
DM_US 33489877-6079259 06l5
The present invention provides, in a first aspect, a stable anti—0:407 antibody
formulation. In one , the formulation comprises a buffer, at least one
stabilizer and an 14137 antibody. In one aspect, a dry formulation comprises one
or more non-reducing sugars and an anti-(14137 antibody, wherein the ratio of non-
reducing sugar to anti—0:407 antibody (molezmole) is greater than 600:1. The
formulation also comprises one or more free amino acids. One or more of the amino
acids also 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 dry, e. g., lyophilized. The
antibody in the formulation may be a full length antibody or an antigen binding
nt f, such as a Fab, Fv, scFv, Fab’ or F(ab')2 fragment.
The formulation can contain any desired non-reducing . In one aspect,
non-reducing sugars that can be included in the formulation include, for example,
mannitol, sorbital, sucrose, trehalose, raffinose, ose, melezitose, dextran,
maltitol, lactitol, isomaltulose, palatinit and combinations thereof. In another aspect,
non-reducing sugars are sucrose, trehalose, ol, and sorbitol. The absolute
amount of non~reducing sugar in the ation is not critical, but the ratio of non—
reducing sugar to anti—(x407 antibody (molezmole) is greater than 400:1 In another
, the ratio of non-reducing sugar to anti-0:407 antibody (molezmole) 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:l, at least about 1400:1, at least about 1500:1, at least about l600:l, at
least about 170021, at least about 1800: 1, at least about 1900: l, or at least about
2000: 1. Generally, it is desirable that the non-reducing sugar is t in an
amount which reduces soluble aggregate ion in a liquid formulation, such as
aggregate formation which occurs upon freezing and thawing and/or drying and
reconstituting. A ratio of non-reducing sugar to anti-0:407 antibody (molezmole)
higher than about 730:1 may give slightly d soluble aggregate formation in
the lyophilized state. The sugarzprotein weight ratio can be greater than 1.5:1 (w/w).
In another aspect, the non-reducing sugar concentrations for liquid (e.g., pre-drying
or post-reconstitution) formulations are in the range from about 10 mM to about 1
M, for example, from about 60 mM to about 600 mM, about 100 mM to about 450
DMAUS 33489877-6 079259 0615
mM, about 200 mM to about 350 mM, about 250 mM to about 325 mM, and about
275 mM to about 300 mM. In r aspect, the amounts of non—reducing sugar in
a dry, (e.g., lized) formulation are in the range from about 40% to about 70%
(w/w of dry formulation). In r aspect, the amounts of non—reducing sugar in a
dry (e.g., lyophilized) formulation are in the range from about 40% to about 60%,
from about 45% to about 55% or about 51% (w/w). In other aspects, the amount of
non-reducing sugar in a dry, (e.g., lyophilized) formulation is greater than about
51% (w/w of dry formulation) when the protein amount is about 31 % (w/w of dry
formulation) or greater than about a 1.611 mass ratio of non-reducing sugar to
protein in the dry formulation. In yet still another aspect, sucrose is the non-
reducing sugar for use in the formulation.
The formulation can contain any desired free amino acid, which can be in the
L—form, the D-form or any desired mixture of these forms. In one aspect, free amino
acids that can be included in the formulation e, for example, histidine, alanine,
arginine, glycine, glutamic acid, serine, lysine, phan, valine, cysteine and
combinations thereof. Some amino acids can ize the proteins against
degradation during manufacturing, drying, lyophilization and/or storage, e.g.,
through hydrogen bonds, salt bridges antioxidant properties or hydrophobic
interactions or by exclusion from the protein surface. Amino acids can act as
tonicity modifiers or can act to decrease Viscosity of the formulation. In another
aspect, free amino acids, such as histidine and arginine, can act as cryoprotectants
and lyoprotectants, and do not llize 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 still yet another aspect, the formulation ns histidine, or histidine and
arginine. In still yet a further aspect, the free amino acid trations for liquid
formulations are in the range from about 10 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 still yet a further , the
amounts of histidine in a dry, (e.g., lyophilized) formulation are in the range from
about 1% to about 10% (w/w of dry formulation), or from about 3% to about 6%
(w/w). In some ments, the amount of histidine in a dry, (e.g., lyophilized)
DMAUS 33489877-6 07925906l5
formulation is greater than about 4% (w/w of the dry ation) when the protein
amount is about 31% (w/w of the dry formulation) or r than about a 0.15:1
mass ratio of histidine to protein in the dry formulation. In still yet another aspect,
the amounts of arginine in a dry, (e.g., lyophilized) formulation are in the
range from
about 4% to about 20% (w/w of dry formulation), or from about 10% to about 15%
(w/w). In some embodiments, the amount of ne in a dry, (e.g., lyophilized)
formulation is greater than about 13% (w/w of the dry formulation) when the protein
amount is about 31% (w/w of the dry formulation) or greater than about a 0.4:1 mass
ratio of ne to protein in the dry formulation. In embodiments of combinations
of amino acids, such as histidine and ne, the molar ratio of total amino acid to
dy ratio can be at least 200:1, about 200:1 to about 500:1, or at least 400: 1.
The formulation can optionally further contain at least one surfactant. In one
aspect, surfactants that can be included in the formulation include, for example,
polysorbate 20, polysorbate 80, a mer (Pluronic®) and combinations thereof.
When present, the surfactant is generally included in an amount which reduces
formation of insoluble aggregates of antibody, e. g., during bottling, freezing, drying,
lyophilization and/or reconstitution. The surfactant concentration, e.g., in a y,
(e.g., lyophilized) or post-reconstitution formulation, is generally from about
0.0001% to about 1.0%, from about 0.01% to about 0.1%, for example about 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08,% or 0.09% (w/V), 0.05% to 0.07% or
0.06% (w/V). The surfactant amount, e.g., in a dry, (e. g., lyophilized) formulation, is
generally from about 0. 01% to about 3.0% (w/w), from about 0.10% to about 1.0%,
for example about 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, or 0.50% (w/w). In
another aspect, the surfactant: antibody molar ratio is about 1:1. The anti—a4B7
antibody can be present in any d amount in the formulation, provided that the
ratio of non—reducing sugar to anti—(x4137 antibody (molezmole) is greater than about
600:1. However, the formulation can contain a high concentration of anti—0L4B7
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/ml, at least about 60 ml/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, from about 40 mg/ml to about 80
mg/ml anti—a4B7 dy, about 60 mg/ml anti-a4B7 antibody. Dry formulations
DM_US 33489877-6079259 0615
(e. g., lyophilized) can contain at least about 5%, at least about 10%, at least about
%, at least about 20%, at least about 25%, at least about 30%, or about 31% or
about 32 % anti—r1407 antibody by .
If desired, the formulation can further comprise a metal chelator and/or an
anti-oxidant, as well as other pharmaceutically acceptable excipients. Suitable metal
chelators include, for e, methylamine, ethylenediamine, desferoxamine,
trientine, histidine, malate, phosphonate compounds, e.g., etidronic acid,
ethylenediaminetetraacetic acid (EDTA), neglycoltetraacetic acid (EGTA),
and the like. Suitable anti-oxidants include, for example, citric acid, uric acid,
ascorbic acid, lipoic acid, glutathione, tocopherol, carotene, lycopene, cysteine and
the like.
The formulation can be a liquid or a solid. Liquid formulations can be
aqueous solutions or suspensions, ed in a suitable aqueous solvent, such as
water or an aqueous/organic mixture, such as water alcohol mixtures. Liquid
ations can have a pH between about 5.5 and about 7.5, between about 6.0 and
about 7.0, or between about 6.0 and about 6.5, such as about 6.0, 6.1, 6.2, 6.3, 6.4 or
6.5. Liquid formulations can be refrigerated (e. g., 2-80C) or frozen (e. g., at -20°C or
—80°C) for storage. Solid formulations can be prepared in any suitable way and can
be in the form of a cake or powder, for example. The solid formulation is prepared
by drying a liquid formulation as described , for example by lyophilization,
spray drying, air drying in a film (e. g., for transderrnal delivery), mixing into a lipid
emulsion and drying as spheres for oral delivery or film for transdermal delivery.
When the formulation is a solid formulation, the formulation can have a moisture
content of no more than about 5%, no more than about 4.5%, no more than about
4%, no more than about 3.5%, no more than about 3%, no more than about 2.5%, no
more than about 2%, no more than about 1.5%, no more than about 1%, or is
ntially anhydrous. Solid ations can be dissolved, i.e. reconstituted, in a
suitable medium or solvent to become liquid suitable for administration. Suitable
solvents for reconstituting the solid formulation include water, isotonic saline,
buffer, e.g., ate—buffered saline, Ringer’s (lactated or se) solution,
minimal essential medium, l/aqueous solutions, dextrose solution, etc. The
amount of solvent can result in a therapeutic protein concentration higher, the same,
DMiUS 33489877-6 0792590615
or lower than the concentration prior to . In one aspect, the reconstituted anti-
a4B7 antibody concentration is the same concentration as in the pre-drying liquid
ation.
The formulation may be sterile, and this can be achieved according to the
procedures known to the skilled person for generating sterile pharmaceutical
formulations suitable for administration to human subjects, prior to, or following,
preparation of the formulation. The formulation can be sterilized as a , e.g.,
before drying and/or after titution by filtration h small pores, through
aseptic processing or by exposure to ultraviolet radiation. Filter pore sizes can be
0.1 urn or 0.2 um to filter microorganisms or 10 to 20 nm to filter virus particles.
Alternatively, or additionally, the dried formulation can be sterilized, e. g., by
exposure to gamma radiation. In one aspect, the anti-a4B7 antibody liquid
formulation is sterilized by filtration before drying.
In one aspect, the formulation is stable upon storage. In another aspect, the
formulation is stable upon storage in the dry state. Stability can be tested by
evaluating physical stability, chemical stability, and/or biological activity of the
antibody in the formulation around the time of ation as well as following
storage at the noted temperatures. Physical and/or chemical stability of a liquid
formulation or a reconstituted dry powder can be evaluated qualitatively and/or
quantitatively in a variety of different ways (see, e. g., Analytical quesfor
Biopharmaceutical Development, Rodriguez-Diaz et al. eds. Informa Healthcare
(2005)), including evaluation of aggregate ion (for example using size
ion (or gel filtration) chromatography (SEC), matrix-assisted laser desorption-
ionization time—of-flight mass spectrometry (MALDI-TOF MS), analytical
ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light
scattering (DLS), multi-angle laser light ring )), flow—based
microscopic imaging, electronic impedance (coulter) ng, light obscuration or
other liquid particle counting system, by measuring turbidity, by y gradient
centrifugation and/or by visual inspection); by assessing charge heterogeneity using
cation exchange chromatography (see also Vlasak and Ionescu, Curr. Pharm.
Biotechnol. 9:468-481 (2008) and Harris et al. J. Chromatogr. B Biomed. Sci.
Appl752z233-245 ), isoelectric focusing (IEF), e.g. capillary technique
DM_US 33489877-607925906l5
, or capillary zone electrophoresis; amino-terminal or carboxy terminal
sequence analysis; mass ometric analysis; SDS-PAGE or SEC analysis to
compare fragmented, intact and multimeric (i.e., dimeric, trimeric, etc.) dy;
peptide map (for example tryptic or LYS—and the like); evaluating biological ty
or antigen binding function of the antibody; and the like. Biological activity or
antigen binding function, e.g., g of the anti-a4B7 antibody to MAdCAM (e.g.,
MAdCAM-l) or inhibition of the binding of a cell expressing u4l37 integrin to
MAdCAM (e.g., -l), e.g., immobilized MAdCAM (e.g., MAdCAM-l),
can be evaluated using various ques available to the skilled practitioner (see
e.g., Soler et al., J. Pharmacol. Exper. Ther. 330:864-875 (2009)).
Stability of a solid-state formulation can also be evaluated qualitatively
and/or quantitatively in a variety of different ways, including direct tests, such as
identifying crystal structure by X-Ray Powder Diffraction (XRPD); evaluating
antibody structure in the solid state using r Transform ed Spectroscopy
(FTIR); and measuring thermal transitions in the lyophilized solid (melting, glass
transition, etc.) using Differential ng Calorimetry (DSC, e.g., to assess
denaturation) and indirect tests such as measuring moisture content by Karl Fisher
test, e. g., to extrapolate the likelihood of chemical instability through hydrolysis.
Measurement of the moisture t of a dry formulation can indicate how likely a
formulation will undergo chemical or physical degradation, with higher moisture
leading to more degradation.
Stability can be measured at a selected temperature for a selected time
period. In one , a dry, (e. g., lyophilized) formulation is stable at about 40°C,
75% 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 (e.g.,
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 (e. g., lyophilized)) is stable at about -20°C for at least about 3
months, at least about 6 months, at least about 9 , at least about 12 months, at
DMAUS 3348987746 079259 0615
least about 18 months, at least about 24 months, at least about 30 , at least
about 36 months, at least about 42 months, or at least about 48 months.
Furthermore, the liquid ation may, in some ments, be stable following
freezing (to, e. g., -80° C.) and thawing, such as, for example, following 1, 2 or 3
cycles of ng and thawing.
Instability may involve any one or more of: aggregation (e.g., non-covalent
soluble aggregation (caused by hydrophobic or charge ctions), covalent soluble
aggregation (e. g., disulfide bond rearrangement/scrambling), insoluble aggregation
(cause by denaturing of the protein at the liquid/air and liquid/solid interfaces)) ,
deamidation (e. g. Asn deamidation), oxidation (e.g. Met oxidation), ization
(e. g. Asp iation), denaturation, clipping/hydrolysis/fragmentation (e. g. hinge
region fragmentation), succinimide formation, N-terminal extension, C-terminal
processing, glycosylation differences, and the like.
A stable formulation can contribute to a low immunogenicity of an anti-a4B7
antibody. An immunogenic anti-(1487 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-0L4B7 antibody can have adverse events (e.
g., site
infusion on) upon treatment or can ate anti-0L4B7 antibody quickly,
resulting in a lower dose than planned by treatment. A report (Feagen et al. (2005)
N. Engl. J. Med. 352:2499-2507) of early study of an anti—0:487 antibody treatment
indicated that human antihuman antibodies developed by week 8 in 44% of treated
patients. The antibody in this study was stored as a liquid and did not contain
polysorbate.
In some embodiments, the formulation can increase the tion of HAHA
negative patients to at least 40%, at least 50%, at least 60%, at least 70%, at least
80% or at least 90% of patients compared to the HAHA results of a less stable
formulation.
In some embodiments, an 1487 antibody formulation has 2 50% major
Charged isoform, 2 55% major charged isoform, or 65 to 70% major Charged
isoform. In other aspects, a stable anti-0:487 antibody ation has 3 45% acidic
charged isofonns, S 40% acidic charged isofonns, S 30% acidic charged isoforms or
22 to 28% acidic isoforms. In still other aspects, a stable :487 antibody
DM_US 33489877—6 0792590615
formulation has 5 25% basic isoforms, 5 20% basic isoforms, 3 15% basic ms,
about 5 % basic isoforms or about 10% basic isoforms. In one aspect, a stable anti-
a4B7 antibody formulation has 2 55% major isoform, 5 30 % acidic isoforms and/or
20% basic isoforms, e.g., as determined by CEX. In another , a stable anti-
a4B7 antibody formulation has 2 50% major isoform, 5 45 % acidic isoforms and/or
< 10% basic isoforms, e.g., as determined by cIEF.
In some aspects, an anti-(1407 antibody dry, solid formulation has 310%
moisture content, S 5% moisture content or < 2.5% re content. The time
required for reconstitution is S 60 minutes, 5 50 minutes or S 40 minutes or S 30
minutes or S 20 minutes.
Monomeric content and/or aggregate content (e.g., as dimers, trimers,
tetramers, pentamers, ers and higher-order ates), i.e., in the liquid
formulation, or in a dry formulation after reconstitution, can be measured by SEC,
MALDI—TOF MS, analytical ultracentrifugation, light scattering (DLS or MALLS),
or nanoscale measurement, such as rticle tracking analysis NTA, NanoSight
Ltd, Wiltshire, UK). tion, characterization and quantification of aggregate
can be achieved in a number of ways, including increasing the length of the SEC
column separation, e. g., by a longer column or by serial attachment of a second or
more SEC column(s) in line with the initial analytical SEC column, supplementing
SEC quantification of monomers with light scattering, or by using NTA.
In one embodiment, an anti-(x457 antibody formulation has 2 90%
monomeric antibody, 2 95% monomeric antibody, or 97 to 99% ric
antibody. In r embodiment, the majority of the material in an anti-0:407
antibody formulation has an average radius of S 20 nm, S 15 nm, S 10 nm, or about
5 to about 7 nm. In one , an anti-0L4B7 antibody formulation has 2 80%
amount heavy plus light chain by protein analysis. In one aspect, there is 2 90%
heavy plus light chain. In another aspect, an anti-(14117 antibody formulation has 5
% aggregate, S 5% aggregate, S 2.5% aggregate S 1.5% aggregate, S 1.0%
aggregate or S 0.5% aggregate. In another , a stable 1407 antibody
formulation has 2 96% monomer and/or 3 2.5% aggregate. In yet another aspect, a
DM_US 33489877607959 0615
stable anti-r1437 antibody ation has about 99% monomer and/or about < 1%
aggregate.
Particle sizes, e.g., of ates or olved excipient, i.e., in
reconstituted formulation can be measured by light obscuration (e.g., liquid particle
counting system (HIAC) by Hach Ultra ics (Grants Pass, OR)), microscopy,
coulter counter, or l (e. g., flow—based) microscopic imaging based system such
as microfluidics imaging (MFI) by Brightwell (Ottawa, CA) or FLOWCAM®
Image particle analyzer by Fluid Imaging Technologies (Yarmouth, ME). In one
aspect, particle size in an anti-a4B7 antibody preparation is about 30 um, about 25
um, about 10 um, about 5 um, about 2 um or 1 um or less. The amount of particles
should be minimized in antibody formulations. In one aspect, the anti~a4B7
antibody formulation has less than 6000 particles 2 10 um and less than 600
particles 2 25 um diameter in one dose (U.S. Pharmacopoeia Chp. 788, light
obscuration counting method; half those amounts by microscopic quantification
). In yet another aspect, an amount of particles
per milliliter, e.g., by MFI
measurement, in a dose of an anti-a4B7 dy formulation, e. g., tituted
formulation is about 500 to about 2000, or about 1000 to about 3000 of 2-10
particles per ml, about 50 to about 350 of 210 um particles per ml and about 0 to
about 50 of 225 um particles per ml.
In one embodiment, an anti-0L4B7 antibody formulation has a binding affinity
of about 60% to about 140% of the reference rd anti—0L4B7 antibody. In one
aspect, an anti-a4B7 antibody in a formulation described herein binds to (14137, e.g.,
on a cell (WO98/06248 or US. Patent No. 7,147,851), at a value of about 80% to
about 120% of the reference standard. In another embodiment, an anti—a4B7
antibody ation has the ability to inhibit at least 50% or at least 60% of the
binding of a cell expressing a4B7 integrin to MAdCAM, e.g., -I, a
MAdCAM-Ig chimera (see U.S. Patent Application Publication No. 20070122404,
also for reference standard examples).
As noted above, freezing of the formulation is specifically contemplated
. Hence, the formulation can be tested for stability upon freezing and thawing.
Accordingly, the antibody in a liquid formulation may be stable upon freezing and
DM‘US 33489877-6 0792590615
thawing the formulation, for example the antibody can be stable after one, two,
three, four, five or more freeze/thaw .
In some embodiments, the formulation is a liquid formulation comprising at
least about 50 mg/ml to about 100 mg/ml 4B7 antibody, a buffering agent (e.
histidine), and at least about 9% (w/w) non-reducing sugar (e.g, sucrose, trehalose or
mannitol). In one embodiment, the ation comprises at least about 50 to about
80 mg/ml, about 60 mg/ml anti-(x4137 antibody, a buffering agent (e.g., histidine), a
free amino acid (e.g., arginine) and at least about 9% or 10% (w/w) non-reducing
sugar (e.g, sucrose, trehalose or mannitol).
In another embodiment, the formulation ses at least about 60 mg/ml
anti-(14137 antibody, a buffering agent (e,g., histidine), a free amino acid (e.g.,
arginine) and at least about 10% (w/w) non-reducing sugar (e. g., sucrose, trehalose
or mannitol). In such embodiments, the buffer concentration is about 15 to about 75
mM, about 25 to about 65 mM, or about 50 mM. The free amino acid concentration
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 dry, solid formulation (e.g., a
lyophilized formulation), comprising a mixture of a non—reducing sugar, an anti-
a4B7 antibody, histidine, arginine, and rbate 80, and the molar ratio of non-
reducing sugar to anti-(1487 antibody (mole:mole) is greater than 600:1.
In another embodiment, the formulation is a dry, solid, amorphous
formulation (e.g., a lyophilized formulation), comprising a mixture of a non-
ng sugar, an anti-(14137 antibody, histidine, arginine, and polysorbate 80, and
the molar ratio of non—reducing sugar to anti—014137 antibody (mole:mole) is greater
than 600:1.
In one embodiment, the ation is a lized formulation comprising
a non—reducing sugar, an anti-(14137 antibody, histidine, arginine and polysorbate 80,
and the molar ratio of non-reducing sugar to anti-(14137 antibody (mole:mole) in the
formulation is greater than 600:1.
In one embodiment, the formulation is a lyophilized ation comprising
a non-reducing sugar, an anti—(x4137 dy, histidine, arginine and polysorbate 80,
wherein the molar ratio of ducing sugar to anti-(x4137 antibody (mole:mole) in
DM_US 33489877—6079259 0615
the formulation is greater than 600:1 and the molar ratio of arginine to anti—(14137
antibody (mole:mole) in the ation is greater than 250:1.
In one ment, the formulation is a liquid formulation and comprises at
least about 60 mg/ml anti-(x4137 antibody, at least about 10% (w/v) non-reducing
sugar, and at least about 125 mM of one or more free amino acids.
In one embodiment, the formulation is a liquid formulation and comprises at
least about 60 mg/ml 14137 dy, at least about 10% (w/v) non-reducing
sugar, and at least about 175 mM of one or more free amino acids.
In one embodiment, the formulation is a liquid formulation and comprises
between about 60 mg/ml to about 80 mg/ml anti-(x4137 antibody, a buffering agent
and at least about 10% (w/w) sugar.
In one embodiment, the formulation is a liquid formulation and comprises
between about 60 mg/ml to about 80 mg/ml anti-a4B7 antibody, ine and at
least about 10% (w/w) sucrose.
In one embodiment, the ation is lized and stored as a single
dose in one vial. The vial is desirably stored at about 2-8°C until it is administered
to a subject in need thereof. The vial may for example be a 20 or 50 cc vial (for
e for a 60 mg/ml dose). The vial may 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 900 mg of anti-(x4137 antibody. In one aspect,
the vial contains about 300 mg of anti~a4B7 antibody,
One or more other pharmaceutically acceptable carriers, ents or
stabilizers such as those described in Remington: The Science and Practice of
Pharmacy, let Edition, Hendrickson, R. Ed. (2005) may be included in the
formulation provided that they do not adversely affect the desired characteristics of
the formulation. Acceptable carriers, ents or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include; additional
buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; ,
chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes);
biodegradable polymers such as polyesters; preservatives; and/or salt-forming
counterions such as sodium.
DM'US 3348987743 0792590615
a4fi7 Antibodies
Anti-014137 antibodies suitable for use in the formulations include antibodies
from any desired source, such as fully human antibodies, murine antibodies, rabbit
antibodies and the like, and any desired engineered antibodies, such as chimeric
antibodies, humanized antibodies, and the like. Antigen—binding nts of any of
these types of antibodies, such as Fab, Fv, scFv, Fab' and F(ab')2 fragments, are also
suitable for use in the formulations.
The anti-(14137 antibody can bind to an epitope on the (14 chain (e.g.,
humanized MAb 21.6 (Bendig et al., US. Pat. No. 299), on the [37 chain (e.g.,
FIB504 or a humanized derivative (e.g., Fong et al., US. Pat. No. 7,528,236», or to
a combinatorial epitope formed by the association of the (14 chain with the [37 chain.
In one aspect, the antibody binds a combinatorial epitope on the (14137 complex, but
does not bind an epitope on the 0L4 chain or the [37 chain unless the chains are in
association with each other. The association of 0L4 in with [37 integrin can
create a combinatorial epitope for example, by bringing into ity residues
present on both chains which together comprise the epitope or by conformationally
exposing on one chain, e.g., the 0L4 integrin chain or the B7 integrin chain, an
epitopic binding site that is inaccessible to dy binding in the absence of the
proper integrin partner or in the absence of integrin activation. In another aspect, the
anti-(14137 antibody binds both the 0L4 integrin chain and the [37 in chain, and
thus, is specific for the (14137 integrin complex. Such antibodies can bind (1407 but
not bind (1413], and/or not bind 0:587, for example. In another aspect, the anti-(14137
antibody binds to the same or substantially the same epitope as the Act-1 antibody
(Lazarovits, A. l. et al., J. Immunol, 133(4): 1857-1862 (1984), Schweighoffer et
a[., J. l, 151(2): 717-729, 1993; Bednarczyk et (11,, J. Biol. Chem, 269(11):
8348-83 54, 1994). Murine ACT—1 Hybridoma cell line, which produces the murine
Act—1 onal antibody, was deposited under the provisions of the st
Treaty on Aug. 22, 2001, on behalf nium Pharmaceuticals, Inc., 40
Landsdowne Street, Cambridge, Mass. 02139, USA, at the an Type Culture
Collection, 10801 sity Boulevard, Manassas, Va. 2209, USA, under
Accession No. PTA—3663. In another aspect, the anti-(x4137 antibody is a human
DM_US 33489877‘6 0792590615
antibody or an (1487 binding protein using the CDRs provided in US. Patent
Application Publication No. 2010/0254975.
In one aspect, the anti-01437 antibody ts binding of 04137 to one or more
of its ligands (e. g. the mucosal addressin, e.g., MAdCAM (e. g., MAdCAM—l),
ctin, and/or vascular addressin (VCAM)). Primate MAdCAMs are described
in the PCT publication WO 96/24673, the entire teachings of which are incorporated
herein by this nce. In another aspect, the anti-014137 antibody inhibits binding
of (14137 to MAdCAM (e.g., MAdCAM-l) and/or fibronectin t inhibiting the
binding of VCAM.
In one aspect, the anti-a4B7 dies for use in the formulations are
humanized versions of the mouse Act-1 antibody. Suitable methods for ing
humanized antibodies are well-known in the art. Generally, the humanized anti-
a4B7 antibody will contain a heavy chain that contains the 3 heavy chain
complementarity ining regions (CDRs, CDRl, SEQ ID N028, CDR2, SEQ
ID N09 and CDR3, SEQ ID NO:10) of the mouse Act-1 antibody and suitable
human heavy chain framework regions; and also contain a light chain that ns
the 3 light chain CDRs(CDR1, SEQ ID NO:1 1, CDR2, SEQ ID NO: 12 and CDR3,
SEQ ID NO:13) of the mouse Act-1 antibody and suitable human light chain
ork regions. The humanized Act—1 antibody can n any suitable human
framework regions, ing consensus ork regions, with or without amino
acid substitutions. For example, one or more of the frame work 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 7x. light chains, and/or the y (e.g., yl ,
y2, 7/3, 74), u, a (e. g., a1, a2), 5 or a heavy chains of human antibodies, including
allelic variants. A particular constant region (e.g., IgGl), variant or portions thereof
can be selected in order to tailor effector function. For example, a mutated constant
region (variant) can be incorporated into a fusion protein to minimize binding to Fc
receptors and/or ability to fix complement (see e.g., Winter at (11., GB 2,209,757 B;
Morrison et al., WO 89/07142; Morgan 61 al., WO 94/29351, Dec. 22, 1994).
Humanized versions of Act-l antibody were described in PCT publications nos.
DMiUS 33489877-6 079259 06 l 5
WO98/06248 and WOW/61679, the entire teachings of each of which are
incorporated herein by this reference.
In another aspect, the anti—(14137 humanized antibodies for use in the
ation comprise a heavy chain variable region comprising amino acids 20 to
140 of SEQ ID N02, 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. Ifdesired, a
suitable human constant region(s) can be present. For example, the humanized anti-
a4B7 antibody can comprise a heavy chain that comprises amino acids 20 to 470 of
SEQ ID N022 and a light chain comprising amino acids 21 to 239 of SEQ ID NO:5.
In another example, the humanized anti-(14137 antibody can comprise a heavy chain
that comprises amino acids 20 to 470 of SEQ ID N02 and a light chain comprising
amino acids 20 to 238 of SEQ ID NO:4. Figure 4 shows an alignment which
compares the generic light chains of human antibodies with murine dies. The
alignment illustrates that the humanized light chain of vedolizumab (e.g., al
Abstract Service (CAS, American Chemical Society) ry number 943609
3), with two mouse residues switched for human residues, is more human than the
light chain of LDP-02 (Figure 3). In addition, LDP-O2 has the somewhat
hydrophobic, e alanine 114 and a hydrophilic site (Aspartate 115) that is
replaced in vedolizumab with the slightly hydrophilic hydroxyl-containing threonine
2O 1 14 and hydrophobic, potentially inward facing valine 115 residue.
Further substitutions to the dy sequence can be, for example, mutations
to the heavy and light chain framework regions, such as a mutation of cine to
valine on residue 2 of SEQ ID NO:14; a mutation of methionine to valine on residue
4 of SEQ ID NO:14; a on of alanine to e on residue 24 of SEQ ID
NO:15; a mutation of arginine to lysine at residue 38 of SEQ ID NO:15; a mutation
of alanine to arginine at e 40 of SEQ ID NO:15; a mutation of methionine to
isoleucine on residue 48 of SEQ ID NO:15; a mutation of isoleucine to leucine on
residue 69 of SEQ ID NO:15; a mutation of arginine to valine on residue 71 of SEQ
ID NO:15; a mutation of threonine to isoleucine on residue 73 of SEQ ID NO: 15; or
any combination thereof; and replacement of the heavy chain CDRs with the CDRs
(CDRI, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) ofthe
mouse Act—1 antibody; and ement of the light chain CDRs with the light chain
DMMUS 334893776079259 O6] 5
CDRs(CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13)
of the mouse Act-1 antibody.
In some embodiments, the anti-a4B7 humanized antibodies for use in the
formulation comprise a heavy chain variable region that has about 95%, 96%, 97%,
98%, or 99% sequence identity to amino acids 20 to 140 of SEQ ID N02, and a
light chain variable region that has about 95%, 96%, 97%, 98%, or 99% sequence
identity to amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132 of SEQ
ID N025. Amino acid sequence identity can be determined using a suitable
sequence alignment algorithm, such as the Lasergene system (DNASTAR, Inc.,
Madison, Wis), using the default parameters. In an embodiment, the anti-a4B7
antibody for use in the formulation is vedolizumab (CAS, American Chemical
Society, Registry number 9436093).
Other (x4137 antibodies may also be used in the ations and dosing
regimes described herein. For example, the (14137 antibodies described in US
2010/0254975 , Inc.), incorporated by reference herein in its entirety, are
le for use in the formulations and s of treating inflammatory bowel
disease in an individual.
The anti-(1437 antibody can be produced by expression of nucleic acid
sequences encoding each chain in living cells, e.g., cells in culture. A variety of
host-expression vector systems may be utilized to express the antibody molecules of
the invention. Such host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified, but also represent
cells which may, when transformed or transfected with the appropriate nucleotide
coding ces, express an anti-a4B7 antibody in situ. These include but are not
limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed
with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding ces; yeast (e.g., romyces, Pichia)
transformed with recombinant yeast expression vectors ning antibody coding
sequences; insect cell systems ed with inant virus expression vectors
(e. g., baculovirus) containing dy coding sequences; plant cell systems infected
with recombinant virus sion vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression
DMJJS 33489877-607925906 I 5
vectors (e. g., Ti plasmid) containing antibody coding ces; or ian cell
systems (cg, COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinant
expression ucts containing promoters derived from the genome of ian
cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter). For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major ediate early gene promoter element from human
cytomegalovirus is an effective expression system for antibodies (Foecking et a1.,
Gene 451101 (1986); Cockett el (1]., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For example, when a large quantity of such a protein is to be produced,
for the generation of pharmaceutical compositions of an antibody molecule, vectors
which direct the expression of high levels of fusion protein products that are readily
purified may be ble. Such s include, but are not limited, to the E. coli
expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the
antibody coding sequence may be ligated individually into the vector in frame with
the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye &
, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol.
2O Chem. 3-5509 (1989)); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione S—transferase
(GST). In general, such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption and binding to matrix hione-agarose beads followed
by elution in the ce of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the cloned target
gene
product can be released from the GST moiety.
In an insect system, Autographa calz’form’ca nuclear polyhedrosis virus
(ACNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding ce may be cloned
3O individually into non—essential regions (for example the polyhedrin gene) of the
virus and placed under control of an ACNPV promoter (for example the polyhedrin
promoter).
DMgUS 33489877-6079259 0615
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the antibody
coding ce of interest may be ligated to an adenovirus transcription/translation
control x, e.g., the late promoter and tite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in vitro or in viva
recombination. Insertion in a non-essential region of the viral genome (e.g., region
E1 or E3) will result in a recombinant virus that is viable and capable of expressing
the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad.
Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for
efficient translation of inserted antibody coding ces. These signals include
the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of appropriate
transcription enhancer elements, ription terminators, etc. (see Bittner et al.,
Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression
of the ed sequences, or modifies and processes the gene t in the specific
fashion desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein ts may be ant for the function of the n.
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the t
modification and processing of the foreign n expressed. To this end,
eukaryotic host cells which possess the cellular machinery for proper processing of
the primary transcript, glycosylation, and phosphorylation of the gene product may
be used. Such mammalian host cells include but are not limited to Chinese hamster
ovary (CHO), NSO, HeLa, VERY, baby hamster kidney (BHK), monkey kidney
3O (COS), MDCK, 293, 3T3, W138, human hepatocellular carcinoma cells (e.g., Hep
02), breast cancer cell lines such as, for example, BT483, H5578T, HTBZ, BT20
DM_US 33489877-6 0792590615
and T47D, and normal mammary gland cell line such as, for example, CRL703O and
The glycosylation machinery of different cell types can produce antibodies
with different glycosylation composition than in another cell type, or no
glycosylation, as with bacterial cells. In one aspect, cell types for production of the
anti-a4B7 dy are mammalian cells, such as N80 or CHO cells. In one aspect,
the mammalian cells can comprise the deletion of an enzyme involved in cell
metabolism and the exogenous gene of interest can be operably linked to a
replacement enzyme, e. g., in a construct or vector for introduction into the cells, e. g.,
by transformation or transfection. The construct or vector with the ous gene
confers to the cells which host the construct or vector a ion age 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 deletion 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, e.g., US. Patent Nos. 5,122,464
or 739).
Solid Formulations
Solid formulations of the invention are generally prepared by drying a liquid
2O formulation. Any suitable method of drying can be used, such as lyophilization or
spray drying. Lyophilization involves freezing a liquid formulation, usually in the
container that will be used to store, ship and distribute the formulation (e.g., a vial).
(See, e.g., Gatlin and Nail in Protein Purification Process Engineering, ed. Roger G.
Harrison, Marcel Dekker Inc., 7 (1994).) Once the formulation is frozen, the
atmospheric pressure is reduced and the temperature is adjusted to allow removal of
the frozen solvent e. g., through sublimation. This step of the lyophilization process
is sometimes referred to as y drying. If desired, the ature can then be
raised to remove any solvent that is still bound to the dry formulation by
evaporation. This step of the lization process is sometimes referred to as
secondary drying. When the formulation has d the desired degree of dryness,
the drying process is concluded and the containers are sealed. The final solid
DMgUS 33489877-6 0792590615
formulation is sometimes referred to as a “lyophilized formulation” or a “cake.”
The lyophilization process can be performed using
any suitable ent. Suitable
lyophilization equipment is available from a number of cial sources (e.g., SP
Scientific, Stone Ridge, NY).
A y of suitable apparatuses can be used to dry liquid formulations to
e a solid (e.g., lized) formulation. Generally, lyophilized formulations
are prepared by those of skill in the art using a sealed chamber that contains shelves,
on which vials of the liquid formulation to be dried are placed. The temperature of
the s, as well as cooling and heating rate can be controlled, as can the
pressure
inside the chamber. It will be understood that various
process parameters discussed
herein refer to ses performed using this type of
apparatus. Persons of ordinary
skill can easily adapt the parameters described herein to other types of drying
apparatuses if d.
Suitable temperatures and the amount of vacuum for primary and secondary
drying can be readily determined by a person of ordinary skill. In general, the
formulation is frozen at a temperature of about -3 0°C or less, such as 40°C
or -
50°C. The rate of cooling can affect the amount and size of ice crystals in the
matrix. Primary drying is generally 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
ature. In one aspect, the primary drying conditions can be set to maintain the
anti-(1407 antibody below the glass transition temperature or collapse temperature of
the formulation. Above the collapse ature, the amorphous frozen matrix
flow (collapse), with a result that the protein molecules
may not be surrounded by a
rigid, solid matrix, and the protein molecules may not be stable in the collapsed
matrix. Also, the formulation can be difficult to fully dry if collapse occurs. The
resulting higher amounts of moisture in the ation can lead to higher rates of
protein degradation and a decrease in the amount of time that the lyophilized product
can be stored before its quality diminishes to unacceptable levels. In one aspect, the
shelf temperature and chamber pressure are selected to maintain the product
temperature below the collapse temperature during primary drying. The glass
transition temperature of a frozen formulation can be ed by methods known
in the art, e.g., by differential scanning calorimetry (DSC). The collapse
DM_US 33489877—6 07925906] 5
temperature can be measured by methods known in the art, e.g. freeze—drying
copy. The ratio of non-reducing sugar to protein (mole:mole) and the
s of other formulation components will impact the glass transition
temperature and collapse temperature. In some embodiments, a glass tion
temperature for an 0:407 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 an 0:407 antibody formulation is about -29°C. In
some ments, the glass transition temperature of an (1487 antibody
formulation is about -30°C, about -31°C, about 32°C, about -33°C, about -34°C,
about -35°C or about -36°C. In some embodiments, a collapse temperature of an
a4B7 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 r embodiment, the collapse temperature of
an a4B7 antibody formulation is about -26OC. Without wishing to be bound by any
particular theory, the faster the ramp—up rate, the higher the collapse temperature of
the product. 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% of the solvent from the anti-(1487 antibody formulation.
Primary drying is dependent on shelf temperature and pressure. The
conditions for primary drying can be determined empirically with lyophilization
under different process parameters. Primary drying may also be mathematically
modeled based on product temperature. Mass and heat transfer equations (Milton, et
al. (1997) PDA J ofPharm Sci & Tech, 51: 7-16), d with knowledge of Rp
and Kv, allow for understanding the combination and interaction of input variables
ing process input variables such as shelf temperature and pressure and
ation variables which are captured in the Rp value. These models can aid in
determining the parameters to be used for an efficient process based on the
limitations of the t temperature by the collapse ature and equipment
capability.
gig _ Am —P.> lnP0= -6l44.96/Tp + 24.0185
dt R
Equation 1 on 2
DMgUS 33489877-6079259 0615
dQ dQ dm
———=AK T—T"(S p) —=AH ————
dz‘ V dz‘ S dt
Eguation 3 Eguation 4
Equation 1 relates the sublimation rate (dm/dt) during primary drying to the
internal cross-sectional area of the ner (AP), the
vapor pressure of ice (PO), the
pressure of the chamber (PC), and an area normalized mass transfer resistance for the
cake and stopper (Rp). PO at the ation ace can be determined from
Equation 2, where P0 is related to the temperature of the product ice at the
sublimation interface, which is an approximation from the product temperature (Tp),
which can be measured with a thermocouple at the bottom of the vial or can be
derived from the ons above when the other variables are determined. Equation
3 relates the heat er rate from the shelf to the vials, where AV is the area of the
vial, Kv is the heat transfer coefficient of the vial, Ts is the temperature of the shelf,
and Tp is the product temperature. Equation 4 couples the heat and mass transfer
equations, where AHS is the heat of sublimation.
As seen from the equations for primary drying, the shelf temperature (T5),
the product temperature (Tp), the chamber pressure (PC), the mass transfer resistance
of the cake (RD), and the heat transfer coefficient (Ky) can affect the sublimation
rate.
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,
e.g., about 2 to 6 hours, about 3 to 5
hours, or about 4 hours, then the shelf temperature is d again to below the
glass transition temperature of the formulation. Annealing can be used to llize
bulking agents and to form larger, more uniform ice crystals. The annealing process
can affect reconstitution time because the annealed, dried cake has a higher surface
area than the unannealed, dried cake. An annealing step of an 0:467 dy
formulation can be at about -30°C to about -1 0°C or about -25°C to about -I 5°C. In
one aspect, an annealing temperature for an a4B7 antibody formulation is about -
200C.
DMfUS 33489877-0079259 0615
Secondary drying is generally conducted at a temperature that is above the
freezing temperature of the liquid formulation. For example, ary 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 ambient temperature, e.g, 20-
°C. The time for secondary drying should be sufficient to reduce the amount of
moisture to <5%.
In another aspect, the lyophilization cycle includes freezing at about -45°C,
annealing at about -20°C, refreezing at about -45°C, primary drying at about -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 by the DP’s thermal
history (freeze, anneal, and refreeze stages) which affects the pore structure of the
cake. The thermal history can also affect the secondary drying stage, where a larger
surface area can aid in desorption of water (Pikal, et al. (1990) Int. J. Pharm., 60:
203-217). Useful process parameters to control during the y and secondary
lyophilization stages can be the shelf temperature and r pressure during each
stage of the drying cycle.
For up, freeze dryer load and solid content can affect the drying cycle.
Primary drying time can be affected by the solids t in the ation. At
higher solids contents, e.g., where overall solids (excipients and/or protein)
concentrations vary more than 10 w/v% or more than 15 w/v%, e.g., 50 to 100%
variance from formulations whose drying time is ined, the drying time can be
affected. For example, a high solids content ation can have a longer drying
time than a low solids content formulation. In some embodiments, the percent usage
of freeze dryer capacity can range from about 25 to about 100%. At higher loading
% of ty, the primary drying time can increase up to 2-fold in comparison to a
lower loading % capacity. The differences between the primary drying times at
different load 0/o increases as the solids content increases. In one embodiment, the
solids content is less than 20—25% and the load is from 25—100%.
Vial size can be ed based on the surface area which will be exposed to
the shelf and to the vacuum during lyophilization. Drying time is directly
proportional to cake height, thus the vial size may be chosen based upon what is
determined to be a reasonable cake height. A vial with a large diameter relative to
DMfUS 33489877!) 079259 0615
volume can provide a high amount of contact with the shelf for efficient heat
transfer during the lyophilization cycle. A dilute antibody solution in a high volume
of liquid will require more time for drying. A balance in vial size versus
formulation volume needs to be struck, e larger vials can be more expensive
to store and ship and have a larger ace to formulation ratio and may expose a
high tion of the formulation to the degradative effects of moisture during long
term e. For a 300 mg dose, anti—(1467 antibody formulation can have a volume
of3 ml, 5 ml, 6 ml, 10 ml, 20 ml, 50 ml or 100 ml prior to lyophilization. In one
, the Vial size is 20 ml for a 60 mg/ml solution in a 300 mg dose.
After lyophilization, the vial can be sealed, e.g., stoppered, under a vacuum.
Alternatively, a gas, e.g., dry air or nitrogen, can be allowed into the vial prior to
sealing. Where oxidation is a concern, the gas allowed into the lyophilization
chamber can comprise a gas which retards or prevents oxidation of the lized
product. In one aspect, the gas is a non-oxygenated gas, e.g., nitrogen, or an inert
gas, e.g., helium, neon, argon, krypton or xenon. In another aspect, the gas is
en or argon.
In some embodiments, the pre-lyophilization anti—(1487 antibody formulation
volume is the same as the pre-administration reconstituted solution volume. For
e, a formulation which is about 5.5 ml ophilization can be reconstituted
to a volume of about 5.5 ml, by adding an amount of liquid, e. g. water or saline, that
takes into account the volume of the dry solids. In other embodiments, it may be
desirable to lyophilize the anti-(1487 dy formulation in a different volume than
the reconstituted solution volume. For example, the anti-a4B7 antibody formulation
can be lyophilized as a dilute solution, e.g. 0.25x, 0.5x, or 0.75x and reconstituted to
1x by adding less liquid, e.g., 75% less, half, or 25% less than the pre-lyophilization
volume. In an embodiment, a 300 mg dose 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-(14137 antibody formulation can
be reconstituted into a more dilute solution than the pre—lyophilized formulation.
Treatment With the Antibody Formulation
DM‘US 3348937743 0792590615
In one aspect, the invention provides a method of treating a disease or
disorder in a subject comprising stering to a subject the anti-a4B7 antibody
formulation described herein in an amount effective to treat the disease or disorder,
e.g., in humans. The human subject may be an adult (e.g., 18 years or older), an
adolescent, or a child. The human subject may be a person 65 years or older. In
contrast to alternative therapeutic dosing regimens, a human subject 65 years or
older does not require any modification of the dosing regimen described herein, and
may be administered the conventional anti—(X4137 dy formulation described
herein.
The t may have had a lack of an te response with, loss of
response to, or was intolerant to treatment with an immunomodulator, a TNF-a
antagonist, or combinations thereof. The patient may have previously received
treatment with at least one corticosteroid (e.g., prednisone) for the inflammatory
bowel disease. An inadequate se to corticosteroids refers to signs and
symptoms of persistently active disease despite a history of at least one 4—week
ion regimen that included a dose equivalent to prednisone 30
mg daily orally
for 2 weeks or intravenously for 1 week. A loss of response to corticosteroids refers
to two failed attempts to taper corticosteroids to below a dose equivalent to
prednisone 10 mg daily orally. Intolerance of corticosteroids includes a history of
Cushing’s syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia and/or
infection.
An immunomodulator may be, for example, oral azathioprine, 6-
topurine, or methotrexate. An uate response to an immunomodulator
refers to signs and symptoms of persistently active disease despite a y of at
least one 8 week regimen or oral azathioprine (21.5 mg/kg), aptopurine
(30.75 mg/kg), or methotrexate (212.5 k). Intolerance of an
immunomodulator includes, but is not limited to, nausea/vomiting, abdominal pain,
pancreatitis, LFT abnormalities, lymphopenia, TPMT genetic 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 to treatment a INF-a nist. A TNF-a
antagonist is, for example, an agent that inhibits the biological activity of TNFo,
DMAUS 33489877-6 0792590615
-46—
and preferably binds INF-(1, such as a monoclonal antibody, e.g., REMICADE
(infliximab), HUMIRA (adalimumab), CIMZIA (certolizumab pegol), SIMPONI
(golimurnab) or a circulating or fusion n such as ENBREL (etanercept).
An inadequate response to a "INF-(1 antagonist refers to signs and symptoms of
persistently active disease despite a history of at least one 4 week induction regimen
of infliximab 5 mg/kg IV, 2 doses at least 2 weeks apart; one 80 mg subcutaneous
dose of adalimumab, followed by one 40 mg dose at least two weeks apart; or 400
mg subcutaneously of certolizumab pegol, 2 doses at least 2 weeks apart. A loss of
response to a TNF—(1 antagonist refers to recurrence of symptoms during
IO maintenance dosing following prior clinical benefit. Intolerance of a TNF-a
antagonist es, but is not limited to infusion related reaction, demyelination,
tive heart failure, and/or infection.
A loss of maintenance of remission, as used herein for ulcerative colitis
subjects, 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—(14137 antibody
ations which (1) can bind (14B7 integrin in vitro and/or in vivo; and (2) can
modulate an activity or function of an (1487 integrin, such as (a) binding function
(e.g., the ability of (1467 integrin to bind to MAdCAM (e. g., MAdCAM-1),
fibronectin and/or VCAM-l) and/or (b) leukocyte ation function, including
recruitment and/or accumulation of leukocytes in tissues (e.g., the ability to inhibit
lymphocyte migration to inal l tissue). In one embodiment, an antibody
in the formulation can bind an (14B7 integrin, and can inhibit binding of the (14B7
integrin to one or more of its s (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-
1, fibronectin), thereby ting leukocyte infiltration of tissues (including
recruitment and/or lation of leukocytes in tissues). In another embodiment,
an antibody in the formulation can bind an (1467 integrin, and can selectively t
binding of the (1467 integrin to one or more of its ligands (e.g., MAdCAM (e.g.,
MAdCAM—I), VCAM—l, fibronectin), y inhibiting leukocyte infiltration of
tissues (including recruitment and/or accumulation of leukocytes in tissues). Such
anti-(1467 dy formulations can inhibit ar adhesion of cells bearing an
(14B7 integrin to vascular endothelial cells in mucosal tissues, including gut—
DM_US 34898776079259.0615
associated s, lymphoid organs or ytes (especially lymphocytes such as T
or B cells) in vitro and/or in vivo. In yet another embodiment, the anti-(14137
antibody formulation of the present invention can inhibit the interaction of (1487
with MAdCAM (e.g., MAdCAM-l) and/or fibronectin. In still yet r
embodiment, the anti-04137 antibody formulation of the present invention can inhibit
the interaction of a4|37 with MAdCAM (e.g., MAdCAM-l) and/or ctin
selectively, e. g., without ting the interaction of (14137 with VCAM.
The anti-a4B7 antibody formulations of the t invention can be used to
modulate (e.g., inhibit (reduce or prevent)) binding function and/or leukocyte (e. g.,
lymphocyte, monocyte) infiltration function of (1487 integrin. For e,
humanized immunoglobulins which inhibit the binding of a4B7 integrin to a ligand
(i.e., one or more ligands) can be administered ing to the method in the
treatment of es associated with leukocyte (e. g., lymphocyte, monocyte)
infiltration of tissues ding recruitment and/or accumulation of leukocytes in
tissues), particularly of tissues which express the molecule MAdCAM (e.g.,
MAdCAM-l).
An effective amount of an anti-a4B7 antibody formulation of the present
invention (i.e., one or more) is administered to an individual (e.g., a mammal, such
as a human or other primate) in order to treat such a e. For example,
inflammatory diseases, including diseases which are associated with leukocyte
infiltration of the gastrointestinal tract (including gut-associated endothelium), other
mucosal tissues, or tissues expressing the molecule MAdCAM (e.g., MAdCAM-l)
(e. g., gut-associated tissues, such as venules of the lamina propria of the small and
large ine; and mammary gland (e. g., lactating mammary gland)), can be treated
according to the present method. Similarly, an individual having a disease
associated with leukocyte infiltration of tissues as a result of binding of leukocytes
to cells (e. g., endothelial cells) expressing MAdCAM (e.g., —l) can be
treated according to the present invention.
In one ment, diseases which can be d accordingly include
inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis,
Celiac disease, nontropical Sprue, enteropathy associated with seronegative
arthropathies, microscopic or collagenous colitis, eosinophilic gastroenteritis, or
DM‘US 33489877-60792590615
pouchitis resulting after proctocolectomy, and ileoanal anastomosis. Preferably, the
inflammatory bowel disease is Crohn’s disease or ulcerative s. The ulcerative
colitis may be moderate to severely active ulcerative colitis. Treatment may result
in mucosal healing in patients suffering from moderate to severely active tive
colitis. Treatment may also result in a reduction, elimination, or reduction and
elimination of corticosteroid use by the patient.
Pancreatitis and insulin-dependent diabetes mellitus are other diseases which
can be d using the formulations of the invention. It has been reported that
MAdCAM (e.g., MAdCAM-l) is expressed by some s in the exocrine
pancreas from NOD (nonobese diabetic) mice, as well as from BALB/c and SJL
mice. Expression of MAdCAM-l was reportedly induced on endothelium in
inflamed islets of the pancreas of the NOD mouse, and MAdCAM-l was the
predominant addressin expressed by NOD islet endothelium at early stages of
insulitis nen, A., et al., J. Clin. Invest, 92: 2509-2515 (1993)). Treatment of
NOD mice with either anti-MAdCAM (e.g., anti—MAdCAMul) or anti B7 antibodies
prevented the development of diabetes (Yang et a1., es, 46:1542—1547
(1997)). Further, accumulation of lymphocytes expressing (1487 within islets was
observed, and MAdCAM-l was implicated in the binding of lymphoma cells via
(14137 to vessels from d islets (Hanninen, A., et al., J. Clin. Invest, 92: 2509—
2515 (1993)) or to the gastrointestinal tract in mantle cell lymphoma (Geissmann et
al., Am. J. Pathol, 01-1705 ).
Examples of inflammatory diseases associated with l tissues which
can be treated using a formulation of the invention include cholecystitis, cholangitis
(Adams and Eksteen Nalure Reviews 6:244—251 (2006) Grant et a[., Hepatology
33: 1065—1072 (2001)), e.g., primary sclerosing cholangitis, Behcet’s disease, e.g., of
the intestine, or pericholangitis (bile duct and surrounding tissue of the liver), and
graft versus host disease (e. g., in the gastrointestinal tract (e.g., after a bone marrow
transplant) (Petrovic et a]. Blood 103:1542-1547 (2004)). As seen in Crohn's
disease, inflammation often extends beyond the mucosal surface, accordingly
chronic inflammatory diseases, such as sarcoidosis, chronic gastritis, e. g.,
mune gastritis (Katakai et 61]., Int. l, 14:167—175 (2002)) and other
thic conditions can be le to ent.
DMgUS 33489877—6079259 0615
The ion also relates to a method of ting leukocyte infiltration of
mucosal tissue. The invention also relates to a method for treating cancer (e.g., an
(14137 positive tumor, such as a lymphoma). Other examples of inflammatory
diseases associated with mucosal tissues which can be treated using a formulation of
the invention include is (mammary gland) and irritable bowel syndrome.
es or pathogens whose etiologies exploit the interaction of MAdCAM
(e. g, MAdCAM-l) with (1487 can be treated with an 1487 antibody in a
formulation described herein. Examples of such diseases include immunodeficiency
disorders, such as caused by human immunodeficiency virus (see e.g.,
W02008140602).
A formulation of the invention is administered in an effective amount which
inhibits binding of 0L4B7 integrin to a ligand thereof. For therapy, an effective
amount will be sufficient to achieve the desired therapeutic (including prophylactic)
effect (such as an amount sufficient to reduce or prevent a4B7 integrin-mediated
binding and/or signaling, thereby inhibiting leukocyte adhesion and infiltration
and/or associated cellular responses). An effective amount of an L4B7 antibody,
e.g., an effective titer sufficient to maintain saturation, e.g., neutralization, of (14137
integrin, can induce clinical response or remission in inflammatory bowel disease.
A ation of the invention can be stered in a unit dose or multiple doses.
The dosage can be determined by methods known in the art and can be ent,
for example, upon the dual’s age, sensitivity, tolerance and overall well-being.
Examples of modes of stration include topical routes such as nasal or
inhalational or transdermal administration, enteral routes, such as through a feeding
tube or itory, and parenteral routes, such as intravenous, intramuscular,
subcutaneous, intraarterial, intraperitoneal, or intravitreal administration. Suitable
dosages for antibodies can 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 3 mg/kg to about 6 mg/kg, or 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.
DMJUS 77-6 0792590615
The final dosage form, e.g., after dilution of the reconstituted antibody (e. g.,
in a saline or 5% dextrose infusion system) of the anti-(1487 antibody can be about
0.5 mg/ml to about 5 mg/ml for administration. The final dosage form may be at a
concentration of between about 1.0 mg/ml to about 1.4 mg/ml, about 1.0 mg/ml to
about 1.3 mg/ml, about 1.0 mg/ml to about 1.2 mg/ml, about 1.0 to about 1.1 mg/ml,
about 1.1 mg/ml to about 1.4 mg/ml, about 1.1 mg/ml to about 1.3 mg/ml, about 1.1
mg/ml to about 1.2 mg/ml, about 1.2 mg/ml to about 1.4 mg/ml, about 1.2 mg/ml to
about 1.3 mg/ml, or about 1.3 mg/ml to about 1.4 mg/ml. The final dosage form
may be at a concentration of about 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.1 mg/ml,
about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6
mg/ml, about 1.8 mg/ml or about 2.0 mg/ml. In one embodiment, the total dose is
180 mg. In another embodiment, the total dose is 300 mg. A 300 mg anti-(1487
antibody dose can be diluted into a 250 ml saline or 5% dextrose solution for
administration.
In some aspects, the dosing regimen has two phases, an induction phase and
a maintenance phase. In the induction phase, the antibody or n—binding
fragment thereof is administered in a way that quickly provides an effective amount
of the antibody or antigen binding fragment thereof suitable for certain purposes,
such as inducing immune tolerance to the antibody or antigen—binding nt
thereof or for inducing a clinical se and ameliorating inflammatory bowel
disease symptoms. A patient can be administered an induction phase treatment
when first being treated by an anti—(1467 antibody, when being treated after a long
absence from therapy, e.g., more than three months, more than four months, more
than six months, more than nine months, more than one year, more than eighteen
months or more than two years since anti—0L4B7 antibody therapy or during
maintenance phase of anti-(1487 antibody therapy if there has been a return of
inflammatory bowel disease symptoms, e. g., a e from ion of disease. In
some embodiments, the ion phase regimen results in a higher mean trough
serum concentration, e.g., the tration just before the next dose, than the mean
steady state trough serum concentration maintained during the maintenance regimen.
In the maintenance phase, the antibody or antigen-binding fragment thereof
is administered in a way that continues the response ed by induction therapy
DMWUS 77-6 0792590615
with a stable level of antibody or n-binding fragment thereof. A maintenance
regimen can prevent return of symptoms or relapse of inflammatory bowel disease.
A maintenance regimen can provide convenience to the patient, e.g., be a simple
dosing regimen or require infrequent trips for treatment. In some embodiments, the
maintenance regimen can include administration of the anti-0L4B7 antibody or
antigen-binding fragment thereof, e. g., in a formulation described herein, by a
strategy selected from the group consisting of low dose, infrequent administration,
dministration and a combination any of the ing.
In one ment, e.g., during an induction phase of therapy, the dosing
regimen provides an effective amount of an anti-a4B7 antibody or antigen-binding
fragment in a formulation described herein for inducing remission of an
inflammatory bowel e in a human patient. In some embodiments, the effective
amount of the anti-a4B7 antibody is sufficient to achieve about 5 ug/ml to about 60
ug/ml, about 15 ug/ml to about 45 ug/ml, about 20 ug/ml to about 30 ug/ml, or
about 25 ug/ml to about 35 ug/ml mean trough serum tration of the anti-0L4B7
antibody by the end of the induction phase. The duration of induction phase can be
about four weeks, about five weeks, about six weeks, about seven weeks, or about
eight weeks of treatment. In some embodiments, the induction regimen can utilize a
strategy selected from the group consisting of high dose, frequent administration,
and a combination of high dose and frequent administration of the anti-(1467
dy or antigen-binding fragment thereof, e. g., in a formulation bed
herein. Induction dosing can be once, or a ity of more than one dose, e.g., at
least two doses. During induction phase, a dose can be stered once per day,
every other day, twice per week, once per week, once every ten days, once every
two weeks or once every three weeks. In some embodiments, the induction doses
are administered within the first two weeks of therapy with the anti—(x467 antibody.
In one embodiment, induction dosing can be once at initiation of treatment (day 0)
and once at about two weeks after initiation of ent. In another embodiment,
the induction phase duration is six weeks. In another embodiment, the induction
3O phase duration is six weeks and a plurality of induction doses are administered
during the first two weeks.
DMiUS 33489877-6 079259 06l5
In some embodiments, e. g., when initiating treatment of a patient with severe
inflammatory bowel disease (e.g., in patients who have failed NFOL therapy),
the induction phase needs to have a longer duration than for patients with mild or
moderate disease. In some embodiments, the induction phase for a patient with a
severe disease can have a duration of at least 6 weeks, at least 8 weeks, at least 10
weeks, at least 12 weeks or at least 14 weeks. In one embodiment, an induction
dosing regimen for a patient with a severe disease can include a dose at week 0
(initiation of treatment), a dose at week 2 and a dose at week 6. In another
embodiment, an ion dosing regimen for a patient with a severe disease can
comprise a dose at week 0 (initiation of treatment), a dose at week 2, a dose at week
6 and a dose at week 10.
In one embodiment, e.g., during a maintenance phase of therapy, the dosing
regimen maintains a mean steady state trough serum concentration, e.g., the plateau
concentration just before the next dose, of about 5 to about 25 pg/mL, about 7 to
about 20 pg/mL, about 5 to about 10 ug/mL, about 10 to about 20 pg/mL, about 15
to about 25 ug/mL or about 9 to about 13 pg/mL of anti-0L4B7 dy. In another
embodiment, the dosing regimen e. g., during a maintenance phase of therapy,
ins a mean steady state trough serum tration of about 20 to about 30
ug/mL, about 20 to about 55 ug/mL, about 30 to about 45 ug/mL, about 45 to about
55 ug/mL or about 35 to about 40 ug/mL of anti-a4B7 antibody.
The dose can be administered once per 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, e. g., once per week, once every 2
weeks, once every 3 weeks or once every 4 weeks can be useful for inducing
remission of active disease or for treating a new patient, e. g., for inducing nce
to the anti- a4B7 antibody. A less nt dose, e.g., 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 preventative therapy, e. g., to maintain remission of a patient with
chronic disease. In one aspect, the treatment regimen is treatment at day 0, about
week 2, about week 6 and every 4 or 8 weeks fter. In one embodiment, the
maintenance regimen es a dose every 8 weeks. In an embodiment, wherein a
patient on a one dose every eight weeks maintenance regimen experiences a return
DM_US 33489877~6 07925906! 5
of one or more disease symptoms, e. g., has a relapse, the dosing frequency can be
increased, e.g., 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 ion in the inflammatory bowel disease of the patient. In some
embodiments, the dosing regimen does not alter the ratio of CD4 to CD8 in
ospinal fluid of patients receiving treatment.
In some aspects, a durable clinical remission, for example, a clinical
remission which is sustained through at least two, at least three, at least four visits
with a caretaking physician within a six month or one year period after beginning
treatment, may be achieved with an optimized dosing regimen.
In some aspects, a durable clinical se, for example, a clinical se
which is sustained for at least 6 months, at least 9 months, at least a year, after the
start of treatment, may be achieved with an optimized dosing regimen.
In one ment, 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 at about six weeks after the initial dose, followed by a
fourth and uent doses of 300 mg every four weeks or every eight weeks after
the third subsequent dose.
In some ments, the method of treatment, dose or dosing regimen
reduces the likelihood that a patient will develop a HAHA response to the anti-a4B7
antibody. The development of HAHA, e. g., as measured by antibodies reactive to
the anti-(14137 antibody, can se the clearance of the anti—a4B7 antibody, e.g.,
reduce the serum tration of the anti-a4B7 antibody, e.g., lowering the number
of anti-(14137 antibody bound to (1467 integrin, thus making the treatment less
ive. In some embodiments, to prevent HAHA, the patient can be treated with
an induction regimen ed by a maintenance regimen. In some embodiments,
there is no break between the ion regimen and the maintenance regimen. In
some embodiments, the induction regimen comprises administering a plurality of
doses of anti—0L4B7 antibody to the patient. To prevent HAHA, t he patient can be
treated with a high initial dose, e.g., at least 1.5 mg/kg, at least 2 mg/kg, at least 2.5
DM‘US 33489877-607925906l 5
mg/kg, at least 3 mg/kg, at least 5 mg/kg, at least 8 rug/kg, at least 10 mg/kg or
about 2 to about 6 mg/kg, or frequent initial administrations, e.g., about once per
week, about once every two weeks or about once every three weeks, of the standard
dose when beginning y with an anti—(x437 antibody. In some embodiments,
the method of treatment maintains at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, at least 90% or at least 95% of patients as HAHA-
negative. In other embodiments, 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
embodiments, the patients, or at least 30%, at least 40%, at least 50% or at least 60%
of patients who develop HAHA maintain a low titer, e. g., 3125, of anti—a4B7
antibody. In an embodiment, the method of treatment maintains at least 70% of
patients as HAHA-negative for at least 12 weeks after beginning therapy with an
anti—(14W antibody.
The formulation may be administered to an dual (e.g., a human) alone
or in conjunction with another agent. A formulation of the invention can be
administered before, along with or subsequent to administration of the additional
agent. In one embodiment, more than one formulation which inhibits the binding of
(1437 integrin to its s is administered. In such an embodiment, an agent, e.g., a
monoclonal antibody, such as an anti—MAdCAM (e.g., anti-MAdCAM-l) or an anti-
VCAM—l monoclonal antibody can be administered. In another embodiment, the
additional agent ts the g of leukocytes to an endothelial ligand in a
pathway different from the d4B7 pathway. Such an agent can inhibit the binding,
eg of ine (C-C motif) receptor 9 (CCR9)—expressing lymphocytes to
thymus expressed chemokine (TECK or CCL25) or an agent which prevents the
g of LFA—l t0 ellular 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 W003/099773 or W004/046092, or anti-
ICAM-l antibody or an oligonucleotide which prevents expression of ICAM, is
administered in addition to a ation of the t invention. In yet another
embodiment, an additional active ingredient (e.g., an anti-inflammatory nd,
such as sulfasalazine, azathioprine, 6-mercaptopurine, 5-aminosalicylic acid
DMAUS 33489877‘60792590615
containing anti-inflammatories, another non-steroidal anti-inflammatory compound,
a steroidal anti-inflammatory compound, or antibiotics commonly administered for
control of [ED (e.g. ciprofloxacin, metronidazole), or another biologic agent (e.g.
TNF alpha nists) can be administered in conjunction with a formulation of the
present invention.
In an embodiment, the dose of the co-administered medication can be
decreased over time during the period of treatment by the formulation sing
the anti-a4fi7 antibody. For example, a patient being treated with a steroid (e. g.
prednisone, prednisolone) at the beginning, or prior to, treating with the anti-(1437
antibody formulation would undergo a regimen of sing doses of steroid
beginning as early as 6 weeks oftreatment with the anti-a4B7 antibody formulation.
The steroid dose will be reduced by about 25% within 4-8 weeks of initiating
tapering, by 50% at about 8-12 weeks and 75% at about 12—16 weeks of tapering
during treatment with the anti—(1467 antibody formulation. In one aspect, by about
16-24 weeks of treatment with the anti-(14137 antibody ation, the steroid dose
can be ated. In another example, a patient being treated with an anti—
inflammatory compound, such as 6-mercaptopurine at the beginning, or prior to,
treating with the anti-(1467 antibody formulation would undergo a regimen of
decreasing doses of anti—inflammatory compound similar to the tapering regimen for
steroid dosing as noted above.
In one embodiment, the method comprises administering an effective amount
of a formulation of the invention to a patient. If the formulation is in a solid, e.g.,
dry state, the process of stration can comprise a step of ting the
formulation to a liquid state. In one aspect, a dry formulation can be reconstituted,
e.g., by a liquid as bed above, for use in ion, e.g. intravenous,
intramuscular or subcutaneous ion. In another aspect, a solid or dry
formulation can be administered topically, e. g., in a patch, cream, aerosol or
suppository.
The invention also relates to a method for treating a disease associated with
leukocyte infiltration oftissues sing the molecule MAdCAM (e. g.,
MAdCAM-l). The method comprises stering to a patient in need thereof an
effective amount ofan anti-04W antibody formulation of the invention. In an
DM_US 33489877—6079259 0615
embodiment, the e is graft versus host disease. In some embodiments, the
disease is a disease associated with leukocyte infiltration of tissues as a result of
g of leukocytes expressing (1437 integrin to gut-associated endothelium
expressing the molecule MAdCAM (e.g., MAdCAM—I). In other embodiments, the
disease is gastritis (e.g., eosinophilic gastritis or autoimmune gastritis), pancreatitis,
or insulin-dependent diabetes mellitus. In yet other embodiments, the disease is
cholecystitis, cholangitis, or pericholangitis.
The invention also relates to a method for treating inflammatory bowel
disease in a patient. In one embodiment, the method comprises stering to the
patient an effective amount of an anti-(14137 antibody formulation of the invention.
In some embodiments, the inflammatory bowel disease is ulcerative colitis or
Crohn's e. In other embodiments, the inflammatory bowel disease is Celiac
disease, enteropathy associated with seronegative arthropathies, microscopic or
collagenous colitis, enteritis (e.g., philic gastroenteritis), or tis.
In some embodiments, treatment with an anti-a4B7 antibody does not alter
the ratio of CD4:CD8 lymphocytes. 8 ratios can be measured in blood,
lymph node aspirate, and cerebro-spinal fluid (CSF). The CSF CD4+:CD8+
lymphocyte ratios in healthy individuals are lly 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 alter the CD4:CD8 ratio to
less than 1.
Articles of Manufacture
In another aspect, the ion is an article of manufacture which contains
the pharmaceutical formulation of the present invention and provides ctions for
its use. The article of manufacture comprises a container. Suitable containers
include, for example, bottles, vials (e.g., dual chamber Vials, a Vial of liquid
formulation with or without a needle, a vial of solid formulation with or without a
vial of reconstitution liquid with or without a needle), syringes (such as dual
chamber syringes, preloaded syringes) and test tubes. The container may be formed
from a variety of materials such as glass, metal or plastic. The container holds the
formulation and a label on, or associated with, the container may indicate ions
DM‘US 3348987745 079259 O6l 5
for use. In another embodiment, the formulation can be prepared for self-
stration 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 repeat
administration (e. g., from 2-6 administrations) of the formulation, e. g., using more
than one portion of a reconstituted formulation. The article of manufacture may
further include other materials desirable from a cial and user standpoint,
including other buffers, diluents, filters, needles, syringes and package inserts with
instructions for use as noted in the previous section.
Clinical and uali Anal sis
In another , the invention is a method for determining that a
pharmaceutical formulation meets product quality standards. The method may
se evaluation of a lyophilized pharmaceutical formulation (e. g., humanized
anti-(1407 antibody) comprising inspecting the formulation to assess appearance,
determining reconstitution time, determining re content of lyophilized
ation, measuring aggregates in lyophilized formulation, measuring
fragmentation, measuring oxidation/deamidation, and optionally assessing biological
activity and potency, wherein achievement of termined standards
demonstrates product is indicated for clinical use.
Acceptable quality levels include g5.0% moisture, 540 minutes
titution time, pH 6.3 :tO.3 of reconstituted liquid, 54.0 to 66.0 mg/ml dy
concentration, 255.0% major isoform by CEX, 296.0% r by SEC, 52.5%
high molecular weight (aggregates), 290% H+L chains by SDS-PAGE, 60 — 140%
of the nce standard on.
The invention will be more fully tood by reference to the following
examples. They should not, however, be construed as limiting the scope of the
invention. All literature and patent citations are incorporated herein by reference.
DEVELOPMENT PROTOCOL FOR MAKING FORMULATION
A. Anti-(14137 Antibody Solution
DMiUS 33489877—607925906l 5
s of frozen, high concentration anti-(1467 antibody preparation
izumab, 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, pH 6.3)
are thawed at room temperature for 16-24 hours. Thawed s are pooled into a
stainless steel compounding vessel and mixed. The ation is then diluted with
Dilution Buffer A (50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, pH
6.3) to 80 mg/mL of vedolizumab and mixed. Sucrose is then added by diluting the
preparation with Dilution Buffer B which contains sucrose (50 mM histidine, 125
mM arginine, 40% sucrose, 0.06% polysorbate 80, pH 6.3). This step dilutes the
anti-(1407 antibody preparation to a liquid formulation of 60 mg/mL vedolizumab,
50 mM histidine, 125 mM arginine, 10% sucrose, 0.06% polysorbate 80, pH 6.3.
B. Lyophilization
Anti-(1407 antibody liquid formulation at 60 mg/ml in 50 mM histidine, 125
mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3 is filled into 20 mL
glass vials with 5.52 mL per vial and the stoppers are placed in the lyophilization
position. Vials are loaded onto shelves set at about 20°C in a lyophilizer. After
loading all vials and closing the door, the shelf temperature is lowered to freeze the
solution, about —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 d to re-freeze the solution, about -45°C. After
equilibration of the vials to this ature, the air is evacuated from the chamber.
When the pressure is 150 mTorr, the shelf temperature is ramped to the primary
drying temperature, about -24°C. Primary drying proceeds until the all of the
crystalline ice has sublimed from the vials. Then the shelf temperature is raised to
27°C for secondary drying for 16 hours, until the moisture is approximately less than
2.5% of the lyophilized formulation. When ary drying is complete, nitrogen
gas is backfilled into the chamber until ambient pressure is reached. The vials are
stoppered and d from the lyophilizer.
C. Storage and Use of lized Anti-(14137 Antibody
Lyophilized vials of anti—(1487 antibody are stored at -70°C, —20°C, 2-8°C or
°C for desired periods of time. When ready for use, a vial is brated to room
DMiUS 3348987760792590615
temperature. Then the contents of the Vial are tituted with a syringe
containing water for injection (“WFI”) using a 21 G needle. The amount of WFI is
determined so the final volume of the reconstituted antibody solution is the same
volume of the pre—lyophilized solution. For a 5.52 ml pre-lyophilization volume, 4.8
ml of WFI is added. The vial is gently swirled and then held for 10-30 minutes to
allow the formulation to reconstitute, then the antibody solution is d using a
syringe and is added and added to an IV bag for IV infusion to a patient.
EXEMPLIFICATION
EXAMPLE 1
COMPARATIVE DATA FOR VARYTNG % SUGAR AND AMINO ACIDS IN
LYOPHILIZED FORMULATIONS
A design of experiments approach was performed to look at the effect of
varying the molar ratio of sugar (sucrose and mannitol) to protein, the molar ratio of
ne to protein, and the molar amount of histidine buffer. Histidine and arginine
are known not to llize during the lyophilization process, making them potential
cryo or lyo protectants. 1.5 mL of formulation was filled into 5 mL vials lyophilized
with Primary Drying at -30°C, 150 mT and ary Drying at 20°C, 150 mT.
The stability of the lyophilized formulations reconstituted to 1.5 ml after different
storage conditions 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 the molar ratio of sugar and arginine was varied. The stability of the
formulation was best at low pH and high molar ratio of (sugar + arginine) to protein.
At the histidine molar amounts ed, histidine did not affect the stability of the
formulation. All formulations had l-2% moisture during storage.
Table 1: Change in Percent Monomer when stored at 5°C, 25°C/60% RH, and 40‘”C/75%
RH for 3 months. t Monomer was measured using Size Exclusion Chromatography
(SEC).
Formulation l “/0 Monomer by SEC
DM7US 3348987743079259 0615
60 mg/mL vedolizumab + l 7
t=0 5°C 25°C 40°C
3 mo 60%RH 75% RH
3 mo 3 mo
———« ‘
mM histidine, 75 mM arginine. 2% sucrose, 98.1 98.1 97.8 96.5 1
0.05"/_o_po_lysorbate 80, RH 6.3
mM histidine. 75 mM arginine. 4% e, 98.0 i 98.2 98.0 97.5
0.05% 01 sorbate 80, H 6.9
m _j__
50 mM histidine, 125 mM arginine, 2% sucrose, 98.0 98.3 98.1 —T 97.4
0.05% polysorbate 80ng 6.7
l _+
50 mM histidine, 125 mM arginine, 4% e, 98.0 98.3 98.1 97.4
0.05% polysorbate SOLQH 6.9
50 mM histidine, 125 mM arginine, 6% e, 98.7 98.4 98.4 98.1
1.5% mannitol, 0.06% polysorbate 80,33 63 L . __4 J
50 mM histidine, 125 mM arginine, 9% sucrose, 98.7 98.3 98.1 98.3
0.06% te 80, RH 6.3
Table 2: Change in t Aggregates when stored 5°C, 25°C/60% RH, and 40°C/75% RH
for 3 months. Percent Monomer was measured using Size Exclusion Chromatography
(SEC).
Formulation % A re ates bx SEC
60 mg/mL vedolizumab + t=0 5°C 25°C 40°C
3 m0 60%RH 75% RH
3 mo 3 mo
mM histidine, 75 mM arginine, 2% sucrose, 0.42 0.53 0.89 1.99
0.05% Bolysorbate 80, PH 6.3
mM histidine, 75 mM arginine, 4% sucrose, 0.41 0.51 0.62 1.15
0.05%Eolysorbate 80, pH 6.9
50 mM histidine. 125 mM arginine. 2% sucrose, 0.42 0.47 0.60 1.23
0.05%jol‘ysorbate 80, pH 6.7 J
50 mM ine, 125 mM arginine, 4% sucrose, 0.36 0.44 0.52 0.82
0.05% polysorbate 801211 6.9 .4
50 mM histidine, 125 mM arginine. 6% sucrose, 0.53 0.49 0.51 0.56 7
1.5% mannitol, 0.06% polysorbate 894.131” 6.3
50 mM histidine, 125 mM arginine, 9% sucrose, 0.51 0.51 0.59 0.56
0.06% polxsorbate 80, pH 6.3
Table 3: Change in Percent Major m when stored at 5°C, 25°C/60% RH, and
40°C/75% RH for 3 months. Major lsoform was measured using Cation Exchange
Chromatography (CEX).
Formulation HO/o jor lsoform lg CEX __~‘ _‘
60 mg/mL zumab + (:0 5°C 25°C 40°C
3 mo 60%RH 75% RH
3 mo 3 mo
i _ T ,.
mM histidine. 75 mM arginine. 2% sucrose, 70.5 68.8 67.4 66.3
0.05%Boljsorbate 80.p1~1 6.3
V“ ,
mM histidine, 75 mM arginine, 4% sucrose, 70.8 98.9 68.0 67.7
H%Bolysorbate 80, pH 6.9 1 .4 J
50 mM histidine, 125 mM arginine, 2% sucrose, 70.5 68.9 67.8 66,5
0.05%polxsorbate 8M1! 6.7 _1
V 50 mM histidine, 125 mM arginlne, 4% ’ P ‘
sucrose, 70.6 68.9 68.0 67.4
0.05% Holysorbate 80. 9H 6.9
1 *
50 mM histidine, 125 mM ne, 6% sucrose. 69.6 69.5 69.3 67.4
1.5% mannitol. 0.06% Bglysorbate 80. pH 6.3
50 mM histidine, 125 mM arginine, 9% sucrose. 69.5 69.3 69.2 68.1
0.06% polysorbate 80, RH 6.3
DM_US 33489877-60792590615
shows the predicted models based on the 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 + )*pH + 0.000516*((moles of sugar+moles
arginine)/moles of protein)). The model for change in percent aggregate per month
at 40°C by SEC analysis is 2.43 - (0.263)*pH - 0.000787*((moles of sugar+moles
arginine)/moles of protein)). The model for change in percent major isoform per
month at 40°C by CEX analysis is -2.54 + (0.109)*pH - 0.00130*((moles of
sugar+moles arginine)/moles of protein)). The center line shows the results for the
tive models and the outer lines show the 95% confidence limit for the
predictive models.
shows alternative models based on the statistical analysis of 40°C
data from Tables 1—3 when the input factors are pH, sugar:protein molar ratio, and
argininezprotein molar ratio. The model for change in percent monomer per month
at 40°C by SEC analysis is -3.02 + (0.370)*pH + 0.000482*((moles of sugar)/(moles
of protein) + 0.000657*((moles of arginine/moles of protein). The model for change
in percent aggregate per month at 40°C by SEC analysis is 2.35 - (0.244)*pH -
27*((moles of sugar)/(moles of protein) — 0.00102*((moles of
arginine)/(moles of protein)). The model for change in t major isoform per
month at 40°C by CEX analysis is -2.92 + (0.210)*pH + 0.00164*((moles of
/)/(moles of protein) — 20*((moles of arginine)/(moles of n)). The
center line shows the results for the predictive models and the outer lines Show the
95% confidence limit for the predictive models.
Example 2
Stability Data
Three primary ity batches of the ation (Batch A, B, and C) were
tested for stability after storage at the prescribed storage condition (5 and 25°C/60%
RH for up to 24 months). All three batches contain the same liquid formulation that
was lyophilized: 60 mg/mL anti-(1407 antibody, 50 mM histidine, 125 mM arginine,
% sucrose, 0.06% polysorbate 80, pH 6.3. For Batch A, 3.5 mL of solution was
DM'US 3348987760792590615
filled into 20 mL Vials and lyophilized, for Batches B and C, 5.52 mL of on
was filled into 20 mL vials and lyophilized.
In a separate study, a single drug formulation of 60 mg/ml anti-(14137
antibody, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06% rbate 80,
pH 6.3 was lyophilized in two volumes, 3.5 ml and 9.5 ml, respectively, to yield
Batches R and S for stability samples, which were analyzed over 38 months. Blanks
are NT (not tested).
The data (Tables 4-19) showed that the antibody formulations ed
stable when stored for up to 38 months at 5°C and up to 30 months at 25°C/60%
RH. All product attributes remained within the specifications h the 38 month
time point.
Table 4: Change in Percent Monomer by SEC when stored at 50C.
fl _1_
Time (months) Batch A Batch B Batch C Batch R Batch S’—I
0 99.8 99.8 99.8 98.9 98.8
1 99.8 99.1 99.2 98.8 99.2
_. 4
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
J g
12 99.4 99.0 99.0
4 98.8 . 98.9
r _J
99.4 99.1 99.1
18 99.5 99.4 L 99.4 98.9 98.9
24 99.4 99.2 99.2 99.0 99.0
‘T _4
99.2
L_ 99.2
38 99.3 99.3
_ T]
Table 5: Change in Percent Aggregates by SEC when stored at 5°C.
T '
kTime (months) BatchA BatchB BatchC BatchR BatchS
T '— ..~ 1
0 T”
0.1 0.1 01 0.2 0.2
T g
0.1 If 0.2 0.2 0.2 0.1
3 0.1 0.2 0.2 T1 0.2 : 0.2
. A :1
6 0.2 0.2 0.2 0.2 0.2
9 0.1 0.2 0.2 0.2
Y i
12 0.2 4
0.2 0.2 0.2 0.2
0.2 0.2 0.2
F“ .
, l
18 0.2 0.2 0.2 0.2 0.2
2 L
24 0.2 0.2 0.2 0.2 0.2
‘ l 1 T
L : 0.2 0.2
- 3 g
38 0.2 0.2
_ I 4
Table 6: Change in Percent Major Isoform by CEX when stored at 5°C.
DMJJS 33489877—6 0792590615
Time (months) Batch A Batch C Batch R Batch 8 I
0 68.6 +__Batch B69.9
1 67.5 68.9
3 68.7 68.8
1 g
6 67.7 68.2
9 70.0 68.3
12 l 67.8 68.3
66.9 67.5
18 67.4 T 67.0
24 68.1 69.6
68.5 .
T '
L I 73.6 73.1 l
Table 7: Change in Percent Acidic Isoforms by CEX when stored at 5°C.
Time (months) Batch A Batch B Batch C Batch R Batch S
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 21.8
12 22.9 21.3 22.1 21.2 21.2
22.7 22.3 T 22.8
18 22.8 22.3 22.6 21.1 21.5
24 21.7 22.1 22.9 20.6 20.7 j
22.8 23.2
38 18.9 19.1
Table 8: Change in Percent Basic Isoforms by CEX when stored at 5°C.
Time (months) Batch A Batch B Batch C Batch R Batch S
0 8.5 9.3 9.1 8.1 7.8
1 10.7 9.4 8.9 7.3 7.7
3 9.7 9.0 8.5 7.6 7.8
R ’
6 9.5 8.7 8.2 7.0 6.7
+ ' 4
9 10.2 9.6 9.3 9.0 8.4
* H g 3 4
12 9.3 10.3 9.9 8.0 7.9
* '
10.4 10.1 9.7
'— T
18 9.8 10.7 10.7 L 7.9 7.7
24 10.2 8.3 l‘
8.1 8.1 8.3
P R
8.7 _: 8.2
l " “g g
38 7.5 7.7
Table 9: Change in % (H+L) by d-SDS Page when stored at 5°C.
Time (months) Batch A Batch B Batch C Batch R Batch S
0 l 98 98 98 96 96
l j
1 98 94 98 98 98
3 F 98 98 98 98 l 98
DM~US 33489871607959.0615
—64-
L l
Table 10: Change in Binding Efficacy when stored at 5°C.
Time (months) Batch A Batch B Batch C Batch R Batch S
. 1
0 Z 107 106 105 93 102
T T
1 106 106 103 103 111
3 101 109 108 91 98
6 97 106 105 114 121
T ?
9 100 93 88 102 102
12 103 T T
101 87 119 116
j l
105 90 94
18 86 101 96 95 104
24 92 82 95 81
1 101
87 94
38 89 91
Table l 1: Change in % Moisture by KF when stored at 5°C
Time (months) l Batch A Batch B Batch C | Batch R Batch S
0 | 0.5 0.60.6
0.5 0.4 06 101.0
L 3i 0.5 0.6 06 l
12 0.6 0.7 4 0.8 1.3
0.6 l 0.6 0.9 0.9
24 I 0.5 0.7 | 0.9 0.9
| l l 30 l 0.7 0. 7
Table 12: Change in t Monomer by SEC when stored at 25°C/60%RH
Time (months) Batch A Batch B Batch C Batch R Batch S
‘ *
0 99.8 99.8 99.8 98.9 98.8
1 99.8 99.1 99.2 98.7 98. 7
3 99.8 99.0 1 99.0 98.6 98.5
6 99.8 99.7 99.7 98.9 989
" f
9 99.0 99.1 99.1 99.1 99.1
T T
12 99.3 98.9 98.9 98.8 98.9
99.3 99.0 99.0
._ .44 __ .
18 99.4 99.3 99.3 98.7 98.9
. T ' ’
24 99.2 L. 99.1 99.1 98.9 98.9
99,0 99.0
DMVUS 334898776079259 06l5
-65—
Table 13: Change in Percent Aggregates by SEC when stored at 25°C/60%RH
Time (months) Batch A Batch B Batch C .1 Batch R Batch S
0.1 0.1 0.1 0.2 0.2
_____..,._
0.2 0.2 0.2 0.2
Table 14: Change in Percent Major lsoform by CEX when stored at 25°C/60%RH
LTimeimonths) Batch A Batch B Batch C Batch R Batch 3
0 68.6 69.9 69.5 71.7 71.6
1 67.2 68.4 68.6 71.2 71.0
3 68.1 68.6 68.2 70.3 70.3
6 65.9 67.8 1 ‘1
67.8 71.5 71.1
9 69.3 67.5 66.3 68.6 69.0
12 66.7 1 67.5 67.4 70.1 70.2
66.2 66.6 ‘T 66.8
18 66.1 65.8 64.9 70.0 70.3
k .
24 66.7 70.1
L L 68.4 68.2 70.6
67.2 67.2
Table 15: Change in Percent Acidic Isoforms by CEX when stored at 0%RH
Time (months) Batch A Batch B Batch C Batch R 1 Batch S
0 22.8 20.8 _1* 21.4 20.3 20.6
1 21.9 21.8 22.2 21.4 21.6
3 21.7 22.2 ““1 22.8 21.8 22.0
1 [
6 22.6 22.9 23.5 21.1 21.4
T— ’
9 19.9 22.1 23.1 21.8 21.8 1
12 23.0 21.4 22.0 21.3 21.3
' T
22.5 22.1 22.7
18 22.6 I 22.1 22.6 21.3 21.5
24 21.7 21.9 22.6 20.7 [ 20.7
”T . '
22.7 23.2
__ 1 1
Table 16: Change in Percent Basic Isoforms by CEX when stored at 25°C/60%RH
Time months) I BatchA Batch B 1 BatchC T Batch R Batch 3
0 8.5 9.3 9.1 8.1 7.8
1 10.8 9.8 9.2 7.4 7.3
3 10.3 t 9.3 9.0 7.8 + 7.7
6 11.5 9.3 8.7 7.4 7.5
DM_US 33489877-60792590615
-66—
.4
11.1
11.2
12.1
.2
Table 17: Change in % (H+L) by Reduced-SDS Page when stored at 25°C/60%RH
Time (months) Batch A f Batch B Batch (3 Batch R Batch 8
l __
0 98 98 98
1 96 96
1 98 ”J 98 98
4 98 98
3 97 98 98 98 T 98
6 97 97 97 97 97
‘ §
9 97 97 97 98 98
fl 4 ‘1
12 98 96 96 98 98
y L
97 97 97
18 r
98 97 97 99 99
24 98 97 22 98 99 99
97
__ 98
Table 18: Change in Binding Efficacy when stored at 25°C/60%RH
Time (months) Batch A Batch B —_I
L Batch C Batch R Batch S
0 107 106 105 93 102
1 115 103
L 109
3 92 113 100 96 94
1 1
6 109 89 97 101 114
9 97 89 85 97 102
P _J
12 83 T
91 123
77
96 91 96
18 106 123 87 92 102
24 103 82 90
J 98 94
84 114
Table 19: Change in % Moisture by KF when stored at 25°C/60%RH
Time s) Batch A Batch B Batch C Batch R 1 Batch S
0 0.5 0.6 0.6 0.8 1.0
1 0.5 0.6 0.5
3 t 0.5 0.7 0.6
6 0.5 0.7 0.7 1.3 1.2
12 0.6 0.8 0.6 0.9 1.0
24 0.7 0.8
L 4 0.6 1.1 1.0
0.8 0.7 +
DMiUS 334898774) 079259 0615
-67—
Cation Exchange Chromatography (CEX)
A phosphate/sodium chloride gradient on a weak cation exchange column is
used in a high performance liquid chromatography system to separate charged
species in anti-(r487 antibody formulations and determine the charge composition of
the antibody species. Acidic Isoforms elute before the Major Isoform and Basic
lsoforms elute after the Major Isoform.
Stability data for all vedolizumab batches generated using a CEX assay is
presented in Tables 3, 6-8 and 14-16. The Tables show, that at these storage
ions, there was no trend of reducing the % Major Isoform below 55.0%.
Size Exclusion Chromatography (SEC)
SEC is med using an analytical SEC column (Tosoh Bioscience, LLC,
King of a, PA). The mobile phase is a phosphatebuffered saline solution 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 listed storage conditions resulted in
ng the % Monomer below 96.0%. rly, the % Aggregates remained
52.5% for all batches at all listed storage conditions.
SDS-PAGE Assay
SDS—PAGE is performed using an Invitrogen (Carlsbad, CA) lycine
gel, 4—20% for reducing condition and 4—12% for non-reducing condition. The
tituted antibody formulation sample is diluted in liquid formulation buffer
then diluted one to two with Tris-Glycine SDS Sample Buffer (2X, Invitrogen)
either with 10% 2-mercaptoethanol (reducing sample buffer) or t 2-
mercaptoethanol (non—reducing sample buffer). Samples are briefly heated and
loaded in comparison with a lar weight marker (Invitrogen). The gels are
stained with colloidal coomassie blue (Invitrogen) according to the manufacturer’s
instruction. Protein bands are analyzed by densitometry to identify the % heavy and
light chain for reduced gels and % lgG for non-reduced gels.
Stability data generated using a Reduced SDS¢PAGE assay are presented in
Tables 9 and 17. No noticeable s were observed for the % Heavy + Light
DMvUS 33489871607939.0615
(H+L) chains at all storage conditions listed for all stability lots. The banding
pattern was similar to that of the nce rd and % (H+L) remained at a level
290%.
Binding Efficacy
HuT78 cells (human T cell lymphoma cells, American Type Culture
Collection, Manassas, VA) suspended in 1% BSA in PBS, 0.01% sodium azide are
contacted with serial dilutions of primary test antibody. After incubation on ice, the
cells are washed and treated with fluorescently labeled secondary dy. After a
further wash, the cells are fixed and suspended in FACS t for analysis by flow
cytometry (Becton Dickinson Franklin Lakes, NJ); also see US. Patent No.
7,147,851.
Binding efficacy of vedolizumab was measured relative to the Reference
Standard and reported as .% Reference Standard and ECSO. Stability data is
ted in Tables 10 and 18. Data for the % Reference Standard showed
variability but remained within the specification limits at all storage conditions. No
evaluated lots of vedolizumab displayed a trend of diminished binding efficacy at
the e conditions listed.
Moisture by Karl Fischer
The ation is ed with methanol for a coulometric Karl Fischer
moisture determination. Moisture data is presented in Tables 11 and 19. All
evaluated lots of vedolizumab had less than 5% moisture at all listed e
conditions.
Capillary lsoelectric ng (cIEFl
cIEF is performed using an iCE280 whole column detection cIEF system
(Convergent Biosciences, Toronto, Ontario). Choice of ampholyte can be as
recommended by the manufacturer or can be a combination of commercially
3O available ampholytes. A useful combination is a mixture of 3-10 and 5—8
PHARMALYTETM (GE Healthcare, Piscataway, NJ).
DMAUS 33489871607959.0615
Example 3: Modeling the Scale-up of the lyophilization process
Quality by design was used while manipulating the load in the freeze dryer
and the solids content of the formulation. The load was varied from 33 to 100%.
The ation solids content was varied from 9 to 27% by including in the loads a
formulation which was 0.5x, 1.0x and 1.5x of the target formulation. These
formulations had similar Tg'. With higher % , the primary drying time
increased. In addition, at higher solids content, the product temperature increased
due to larger RP. The load also has an effect on both stages of drying (.
Example 4: Non-Clinical Safety Study
A study was designed to e the effect of natalizumab and vedolizumab
on immune surveillance of the CNS in Rhesus EAE. Eight animals were dosed with
a placebo control, once . Seven animals were dosed at 30 mg/kg, once
weekly, with natalizumab. Seven s were dosed at 30 mg/kg, once weekly,
with vedolizumab. The clinical symptoms of EAE were observed; the frequency
and ratio of leukocyte subsets in CSF were measured by flow cytometry; the total T2
lesion load in the brain was measured using MRI; and lesion load and demyelination
of the brain was measured using histopathology.
Vedolizumab did not delay onset of clinical symptoms of EAE as compared
to placebo control. It did not inhibit the incidence of EAE, nor the magnitude of
clinical scores. Natalizumab significantly 5) d the onset of clinical
symptoms of EAE as compared to o control. It inhibited the incidence of
EAE and the magnitude of clinical scores. (
Vedolizumab did not prevent infiltration of the CSF by leukocytes, T
cytes (helper T lymphocytes, cytotoxic T lymphocytes), B lymphocytes,
natural killer cells, or monocytes. In st, natalizumab inhibited infiltration of
the CSF
Vedolizumab did not inhibit the accumulation of brain lesions, as detected by
increased T2 and decreased MTR values via MRI. Natalizumab prevented lesion
formation in all but one animal, Significant (p<0.05) tion in brain infiltrates
and demyelination was measured by histology.
DM‘US 33489877—607925906] 5
The (1487 integrin was ted by vedolizumab during the investigation, as
shown by a itive binding assay between vedolizumab dosed in vivo and an
analytical anti-(14W onal antibody added ex vivo. The analytical anti-(1407
mAb does not bind to memory helper T cytes in animals dosed with
vedolizumab. The lack of effect of vedolizumab in the CNS is therefore due to the
gastrointestinal-tropic biology of the (14137 integrin.
In summary, vedolizumab (an (1407 antagonist) does not inhibit EAE. In
st, natalizumab 1 and (14137 antagonist) does inhibit EAE. The (14131
integrin mediates infiltration of the CNS in EAE. Thus, vedolizumab may have a
lower risk of predisposing patients to PML than natalizumab because it does not
antagonize the (1401 integrin and impair immune surveillance of the CNS in Rhesus
EAE.
e 5: Phase I Clinical Study 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 sodium chloride, 0.05% polysorbate 80, pH 6.0 (stored
long term -70°C and up to 3 months at -20°C)) and 10 subjects ed 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
aphic or baseline characteristic. Mean age ranged from 35.4 to 51.0 years;
individual subject ages ranged from 21 to 63 years.
PK Results
Vedolizumab was administered as a 30-minute intravenous infusion of 0.2 to
.0 mg/kg. The Cmax and area under the serum drug concentration-time curve of
(AUC) values increased with increasing dose. The dose-corrected Cmax was
approximately the same across s, indicating dose proportionality for this
parameter. The dose—normalized area under the serum drug concentration value
from time zero to infinity (AUC0_m{) increased with increasing dose up to 2.0 mg/kg,
DMJJS 33489877—6 079259 0615
indicating there was a nonlinear increase in eith increasing dose over the
lower range of doses administered in this study. fter, AUCo-mfincreased
proportionally with dose, indicating linearity of AUCMnfover the dose range 2.0 to
.0 mg/kg. The increase in AUCO_mf‘WaS approximately 24-fold higher than
expected at the 10.0 mg/kg dose ed with the 0.2 mg/kg dose.
Similarly, estimates of clearance, volume of distribution, and terminal half-
life were dose-dependent over the dose range 0.2 to 2.0 mg/kg. As dose increased,
clearance was reduced, bution volume increased, and, consequently, the
terminal elimination ife was prolonged. However, from 2 to 10.0 mg/kg, there
was no apparent change in these parameters, which suggests a saturation of a rapid
elimination process for vedolizumab at low concentrations. Slower linear
elimination processes likely account for a large on of clearance of vedolizumab
at higher doses.
In some subjects who developed HAHA to vedolizumab, a faster clearance
of vedolizumab was observed as compared to the HAHA-negative subjects within
the respective dose level.
Table 20: Overview of zumab PK by Dose Cohort Following IV
Administration of 02-100 mg/kg Vedolizumab in Healthy Subjects (PK Analysis
Set)
Parameter VDZ N Mea SD Geometri %C Media ~1_Min_1 Max—
dose n cMean V n
Cnm ‘02 ””4 5.65 0.62 ‘562 11.1 5.45 5.13 6.56
(pg/mL) mg/k 9
0.5 4 ‘106 ‘209 ‘104 .197 ‘106 *807 13.1
mg/k
2.0 7 59.3 11.6—1 58.4 l19.6“58.4 ’47.6 ”78.4
mg/k
DMVUS 33489877—6079259 06l 5
6.0 6 151 19.1 150 12.6 157 120 168
.0 7 1 22.]—1 243 9.07 242 213 4‘281
AUCMW 0.2 4 31.6 4.98 31.3 15.8 31.6 25.7 37.5
(day* ug/mL mg/k
) g
0.5 4 127 48.0 119 37.9 129 70.9 178
mg/k
2.0 7 964 147 955 15.27972 772 1170
6.0 6 3090 749 3020 24.2 2830 2360 4100
mg/k
.0 7 4870 624 4840 12.8 4750 4120 5870
mg/k
AUCO-mf 0.2 4 39.5 5.79 39.1 14.7 40.2 131.7 45.7
(day*11g/mL mg/k
0.5 4 134 T48.91127 36.5 ‘134 79.2 188
mg/k
2.0 7 979 146 T969 14.9 r993 784 1180
mg/k
”6.0 673100 750 3030 24.2 2840 239077 4110
DMiUS 3348913776 0792590615
W _* 1——~——r———-———
.0 7 4880‘637‘14850 4750 T———,L———~4130 5920
mg/k
”Pg _. L 1
.2 W
VZ(L) 0.2 4 4.02 0.15 4.02 3.76 4.03 3.83 4.18
mg/k 1
0.5 4 4.92 0.62 4.89 12.6 4.66 4.52 5.84
mg/k 0
~— L
2.0 7 3.34 0.66 3.28 19.9 3.23 2.29 4.27
mg/k 5
6.0 “6 2.98 0.64 2.92 21.6 2.98 20613.98
mg/k 4
.0 7 2.89 1.02 2.73 35.2 2.98 1.49 4.58
mg/k
CL(L/day) 0.2 4 0.41 0.04 0.412 10.1 0.395 0.38 0.47
mg/k 3 2 8 6
0.5 '4‘0.31"‘0.10fl0.297 74.3 0.291 *021 0.44
mg/k 0 6 2 6
2.0 7‘0.1’6"‘0.01 0.164 10.7 0.162 0.14 0.19
mg/k 5 8 5 4
6.0 “‘6 0.14 0.03 0.136 22.0 ”0.145 0.08 0.16
mg/k O 1 3 6
DMJJS 33489877-6 0792590615
.0 r‘7’T0.1410.0”2'T0139 LO.135 0.10 0.17"
rug/k 0 4 3 1
t1/2(day 0.2 4 6.7“““073 T6.76 fl10.8 T“6—95 5.79 7.47“
mg/k 6
0.5 4 11.7 ”2.83 11.4 ”T242 11.4 W
mg/k
2.0 7T14.1 39 18.9 14.3 10.6 17.5
mg/k
6.0 6 15.1 3.15 14.8 20.9 140 1194203
mg/k
.0 7 14.8 7.38 13.7 49.81125 8.26”"507
mg/k
L _1
Abbreviations: AL'C0-mf=area under the drug concentration—time curve,
extrapolated to infinity; AUCMasF area under the drug concentration-time curve
from administration time to the last measurement time point at which the
concentration is above the lower limit of quantification; CL=total clearance;
Cmafimaximum drug concentration; t1 /2=terminal half-life; me of
distribution based on the terminal phase.
After reaching Cmax, serum concentrations of Vedolizumab fell in a generally
monoexponential fashion until concentrations reached approximately 1 to 10 mg/L.
Thereafter, concentrations appeared to fall in a ear fashion.
The Cmax and AUC values increased with increasing dose. For the available
data, the dose—corrected Cmax was approximately the same across cohorts,
indicating dose proportionality for this ter. The dose—normalized AUCO_mf
increased with increasing dose up to 2.0 mg/kg, indicating there was a nonlinear
increase in AUCO-inf with sing dose over the lower range of doses administered
DMgUS 33489877—6.079259.06l5
in this study. Thereafter, AUCO-jnf increased tionally with dose, indicating
ity of AUCO—inf over the dose range 2.0 to 10.0 mg/kg. The increase in AUCO-
infwas approximately 24-fold higher than expected at the 10.0 mg/kg dose
compared with the 0.2 mg/kg dose.
PD results
The PD parameters of Vedolizumab following a 30-minute intravenous
infusion of 0.2 to 10.0 mg/kg vedolizumab by cohort are summarized in Table 21
and Table 22 for Act-1 and MAdCAM respectively.
Table 21: ew of Vedolizumab Pharmacodynamics, Percent Inhibition
of O/OAot-1+ [CD4+ CD45Roh‘gh], by Dose Cohort ing 1v Administration of
0.2-10.0 mg/kg Vedolizumab in Healthy Subjects (PD Analysis Set)
Parameter VDZ I
N Mean SD Geometri %C Media Min Max
dose cMean V 11
EM 0.2 4T99.6 0.38 99.6 0.38 99.6 99.1 100
(%Inhibition) mg/k 7 8
0.5 4 99.5 l"6.59 99.5 T"0.60 "99.5 98.9 1700
mg/k 9 2
2.0 6 99.9 0.17 99.9 10.17 100 99.6 100
mg/k 2 2
‘ l
6.0 6 T100 '0.00 ””100 0.00 “100 100 100
mg/k 0 0
.0 6 T997 032 '997 0.32 99.8 T993 i100
mg/k 6 7
AUECW 0.2 44 ‘4030 m 3920 "25.2 ”4090 2760 T5160
(%lnhibition*d mg/k
) 8
J * g
DM-US 33489877-60792590615
0.5 4 6430 1450 6300 22.6 6530 4860 7810
mg/k
2.0 6 1320 623 13200 4.72 12900 1280 1450
mg/k 0 0 0
e ————1 q 1
6.0 6 1670 3030 16500 18.1 16300 1330 2010
mg/k 0 0 0
l 4
r a... a
100 6 1930 644 19300 3.33 19600 1820 1990
mg/k 0 0 0
AUECmnFarea under the drug effect versus time curve from time 0 to the
time of the last non-zero concentration; Emax=maximum drug effect
Table 22: Overview of Vedolizumab Pharmacodynamics, Percent Inhibition
of %MADCAM+ [CD4+ hig‘h], by Dose Cohort ing 1v
Administration of 0.2-l0.0 mg/kg Vedolizumab in Healthy Subjects (PD Analysis
Set)
2 ‘
Parameter voz N Mean SD Geometri '%C Media Min Max
dose cMean V 11
EW ‘02 4 99.2”‘353 ”99.2 0.54 99.4 98.4 99.6
(%lnhibition) mg/k 7 2
0.5 4 99.6 L032 99.6 0.32 99.5 99.3 100
mg/k 3 4
2.0 6 99.7“036 99.7 0.36 99.7 99.2 100
mg/k 5 6
6.0 6””9‘9‘8 0.27 ‘998 0.28 l100 99.4 W
mg/k 9 0
10W6 100 “000" 100 0.00 100 ”100 100
mg/k 0 0
DMfiUS 334898774) 079259 0615
g T
WA ‘ *4216 "3160 T4440
(%Inhibition*d mg/k
) g
i _l______
”0.5 4T6770 1400l6660 *206 6840 5170 8230
mg/k
r2.0 ”‘6” 1300 796 “13000 6.12 *13000 #1170 l7390
mg/k 0 0 0
6.0 ’6 1620 13320 '15900 +20.5 15800 1180 2000
mg/k O O 0
.0 6 1770 1330 17700 7.5 17700 1650 1900
mg/k 0 0 0
L. _1__J
AUECo-mf=area under the drug effect versus time curve from time 0 to the
time of the last non-zero concentration; Emax=maximum drug effect
Vedolizumab inhibited the PD parameters, Act-1 and -l—Fc,
nearly maximally at all time points where vedolizumab was able in serum.
Once vedolizumab concentrations sed below the limit of detection of the
assay, the inhibition of Act-1 and MAdCAM~1~Fc returned to approximately the
baseline level.
In some subjects who developed HAHA to zumab, a faster loss of
014B7 receptor saturation was observed as compared to the HAHA-negative subjects
in the respective dose level.
Safety Results
Vedolizumab was generally safe and well tolerated at 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.
DMWUS 33489877—6 07925906 I 5
Immunogenicity/Human Antihuman Antibody (HAHA) ion
One (10%) subject in the placebo group and 21 (54%) subjects in the
combined zumab dose groups had a positive HAHA at some point during the
study. Although positive HAHA samples were observed in all dose cohorts, HAHA
titers >125 were found only in the 2 lowest vedolizumab dose groups. Dose-
dependent suppression of HAHA formation has been ed previously with
vedolizumab. Nineteen of the 22 vedolizumab-treated subjects who were HAHA-
positive had neutralizing HAHA present.
Table 23: Overview of Human Antihuman Antibodies Findings: Safety
Population
rPlacebo 0.2 20.5 12.0 6.0 10.0 Combined—l
N=10 mg/kg mg/kg mg/kg mg/kg mg/kg VDZ
VDZ VDZ VDZ VDZ VDZ N=39
N=8 N27 N=8 N=8 N=8
Subjects r10 8 7 8 8 48— 39
Tested
AnyHAHA-TUO) 6(75) ”11(57) 2(25) 3(38) 6(75) 121(54)
Positive,n
0%»)
Highest 1(10) 5(50) 2(29) 4—2725) 3(38) +605) 17(44)
HAHA
Titer<125,
n(°/o)
t 0 2(25) 2(29) 0 0 0 +400)
HAHA
Titer2125,
n(%)
”Any T 5(63) 7(57) 2(25) “3(38) “5(63) )
Neutralizing
HAHA
Positive,
n(%)
"Highest 0 ‘3(33) T2(29) ‘2(25) “1‘3 (38) T5(63) T1588)
Neutralizing
HAHA
Titer<l25,
DMgUS 3348987746 079259.06] 5
Highest
Neutralizing
HAHA
TiteerZS,
n(%)
One t in the placebo group and 11 subjects in the vedolizumab group
were tently HAHA-positive.
Table 24: Overall Human Antihuman Antibody Status (Safety Population)
Placebo 0.2 mg/kg 05 mg/kg 2.0 mg/kg V6.0 mg/kg 10.0 Combined
N=10 VDZ VDZ VDZ VDZ mg/kg VDZ
N=8 N=7 N38 N=8 VDZ N=39
‘L L.-
HAHA 9 (90) 2 (25) 3 (43) 6 (75) 5 (63) —_l 2 (25) 18 (46)
negative3
n(%)
Isolated 0 2 (25) 1 (14) 1 (13) 1 (13) 5 (63) 10 (26)
HAHA"
n(%)
PerSIStent. 1 (10)_ 4 (50) 3 (43) 1 (13) 2 (25) 1 (13) 11 (28)
HAHA'2
n(%)
a HAHA Negative: Subjects with no positive HAHA results
b Isolated HAHA: ts with only 1 positive HAHA sample with titer
c Persistent HAHA: Subjects with 2 or more positive HAHA samples, or 1
positive sample with titer Z25
Conclusions
This phase 1 study characterized the PK/PD and initial safety profiles of
vedolizumab derived from CHO cells. The results of this study were used to support
dose ion for phase 3 pivotal trials in inflammatory bowel disease.
zumab demonstrated dose proportionality over the tested dose range
for the Cmax parameter; however, dose—dependent changes in AUCO-inf, CL, Vz,
DM_US 33489877-6079259 0615
and t1/2 were observed from 0.2 to 2.0 mg/kg, suggesting ear PK behavior of
zumab. At dose levels greater than 2.0 mg/kg, no further changes in these
parameters were observed, which suggests a saturation of a rapid elimination
process for vedolizumab at low concentrations. Slower linear elimination processes
likely account for a large fraction of clearance of vedolizumab at higher doses.
Vedolizumab inhibited the PD parameters, Act-1 and MAdCAM-l-Fc, at or
near maximal levels at all time points when vedolizumab was measurable in serum.
Once vedolizumab concentrations decreased below the limit of detection of the
assay, the inhibition of Act-1 and MAdCAM—l—Fc returned to approximately the
baseline level.
In some subjects who ped HAHA to vedolizumab, a faster clearance
of zumab and loss of (14137 receptor saturation was observed as compared to
the HAHA—negative subjects within the respective dose level.
Vedolizumab was well—tolerated. No deaths, SAEs, or AEs leading to
tinuation of study drug administration ed during the study, nor were any
dose-toxicity relationships observed. No systemic unistic infections
(including PML) or neoplasms were reported.
Unlike nonspecific (14 antagonists, vedolizumab was not associated with
lymphocytosis or mean increases in circulating eosinophils, basophils, or
monocytes, nor was there any ce of depletion of lymphocytes.
Vedolizumab did elicit the formation of HAHA, but the highest titers (>125)
were observed only in the 2 lowest dose groups, a finding that supports previous
observations of a dose—dependent reduction in immunogenicity. These data show
that the administration of higher doses of zumab may minimize clinically
significant HAHA formation.
In conclusion, vedolizumab was generally safe and well tolerated when
administered in single doses of 0.2 to 10.0 mg/kg to healthy subjects.
Example 6: Determination of the effect of vedolizumab on the 8
3O ratio
DMVUS 33489877—6 079259 0615
Healthy subjects ages 1845 were d with a single 450 mg dose of
vedolizumab reconstituted from a lyophilized formulation of 10% sucrose and
diluted into an on system of 0.9% saline. Cerebrospinal fluid (CSF) was
collected by lumbar puncture before (baseline) and 5 weeks after the single 450—mg
dose of vedolizumab. Each subject served as his/her own control.
A 5-week time point was selected based on a previous study that showed
patients with MS 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. Miller et al. NErzgl JMed. 2003;348(1):15-23); and also e
at 5 weeks, a 450—mg dose of zumab is sufficient to saturate the target and
provides serum concentrations that exceed estimated steady-state trough levels
associated with the phase 3 dose regimen of 300 mg every 4 weeks.
Approximately 15 mL CSF was obtained from each t for
immunophenotyping. CSF samples were included for analyses if they met the
following criteria: £10 RBCs/uL per sample (to minimize peripheral blood
contamination); negative CSF culture result; adequate T-lymphocyte numbers in
each flow cytometry ; and no detection of serum antibodies to vedolizumab.
Week 5 median (34.80 ug/mL) and dual subject serum vedolizumab
concentrations (range 24.9—47.9 ug/mL) were higher than projected steady-state
trough concentration (~24 ug/mL) for the phase 3 dose regimen. A high degree
(>90%) of (14137 receptor saturation was observed at week 5 as ed by
MAdCAM-l-Fc, indicating vedolizumab’s saturation of its target at the time of
endpoint assessment.
Vedolizumab was not detected in any CSF sample tion limit = 0.125
its/mm-
Effect on CD4+ and CD8+ T ijphocyte Numbers and Ratio
Vedolizumab did not cantly reduce CD4+:CD8+ ratio (Table 25).
None of the subjects had a postdose CD4+:CD8+ ratio <1 (p < 0.0001 (l—sided 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 CSF % CD4+
DMiUS 33489877-6079259 0615
and °/o CD8+ T lymphocytes (Table 26). Also, no significant changes in peripheral
blood WBC, CD4+ and CD8+ memory T lymphocytes (Table 27) were observed.
Table 25: Effect of Treatment on CSF D8+ Ratio (Evaluable
Population, n=13)
Baseline Week 5 TCD4+zCD8+ Ratio
Difference'l
D8+ ratio 3.59 (0.273) 3.60 (0.265)* L001 (0197)
Mean (SE) Range 1.53-5.67 1.42—5.15
90% 2-sided CI for 300.419 3.132, 4.077
ratio
90% 2—sided CI for -0.337, 0.363
difference
fidence interval
*p<0.0001 (one sided one sample t—test for H02u<1 vs H1: u>=1).
TDifference is defined as week 5 ratio minus baseline ratio
Table 26: Treatment Effect on CSF CD4+ and CD8+ Lymphocyte Count
(Evaluable Population, n=13)
Baseline Week 5
CD4+ as 0/0 of 75.160 (7.3831) 74.215 (6.3732)
Lymphocytes, mean (SD)
CD8+ as % of 22.272 (5.4320) 22.007 (6.1624)
Lymphocytes, mean (SD)
Table 27: Peripheral Blood Memory T Lymphocytes (RO+) Counts‘
(Evaluable tion, n=13)
l Baseline Week 5
1 Mean (SD) Mean (SD)
DMkUS 33489877-6 079259 0615
CD4+CD45RO+ ] 27.85 (4.98) ] 27.06 (5.02)
45RO+(%) l 11.24 (3.40) £0.78 (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
subjects had a reduction in the post-dose CSF CD4+:CD8+ ratio to less than 1.
Vedolizumab was not detected in CSF. In addition, there was no change observed in
the total WBCs or memory T lymphocyte CD4+ and CD8+ s in peripheral
blood. Saturation of the target 7) in blood occurred in all subjects at the time of
endpoint assessment. The CSF CD4+ and CD8+ lymphocyte levels and ratio were
similar to those previously reported in the literature.
These results are consistent with vedolizumab’s lack of effect on both
physiologic CNS immune surveillance and ogic CNS inflammation of
s (See Example 4).
Example 7: Long-Term Clinical Experience with Vedolizumab for the
Treatment of IBD
A phase 2 open-label safety extension study was completed to assess the
long—term cokinetics (PK), pharmacodynamics (PD), safety, and efficacy of
vedolizumab. Patients were aged 18 to 75 years old, and had either previously
participated in an earlier PK/PD/safety study in ulcerative colitis patients or had IBD
symptoms for at least 2 months ed endoscopically and/or histopathologically
and/or ogically within 36 months of screening.
All patients received an intravenous dosing regimen of either 2 mg/kg or 6
mg/kg ofvedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125 mM
sodium chloride, 0.05% polysorbate 80, pH 6.0 (stored long term -70°C and up to 3
mo -20°C)) on days 1, 15 and 43, followed by a dose every 8 weeks for up to a total
of 78 weeks. Patients were either treatment-naive ulcerative colitis or Crohn’s
disease patients, or ulcerative s patients that had participated in an earlier
clinical trial.
DM_US 33489877-60792590615
Efficacy/quality of life (QoL); partial Mayo score (PMS), Crohn’s e
activity index (CDAI), and Inflammatory Bowel Disease Questionnaire (IBDQ)
were used to assess the results of the study.
PK Results
Mean pre-infusion vedolizumab concentrations were dose proportional, and
remained steady and detectable throughout the study.
PD Results
Receptors (%ACT-1 + [CD4+CD45RO HIGH] and °/o MADCAM+
[CD4+CD45RO HIGH] were almost fully inhibited hout the study period at
all dose levels.
Partial Mayo Score
Baseline mean PMS was higher for treatment-naive ulcerative colitis patients
(5.4) than for ulcerative colitis rollover patients (2.3). By day 43, mean PMS
showed a nced decrease for both rollover and treatment-naive ulcerative
colitis patients. By day 155, mean scores of the two groups were similar. Mean
PMS continued to decrease through day 267, and leveled off thereafter.
s e Activity Index
CD patients’ mean CDAI decreased from 294.6 at baseline to 237.7 at Day
43, and continued to se through day 155 (156.1).
IBDQ
Ulcerative colitis rollover patients had the highest mean IBDQ scores at
baseline. By day 43, mean IBDQ scores had increased in all three disease groups.
Mean IBDQ scores continued to increase over time in all 3 disease groups, reaching
a maximum at day 155 for Crohn’s Disease patients, and at day 491 for ent-
na’r’ve ulcerative colitis patients and ulcerative colitis rollover patients.
C- ve protein
DM‘US 33489877—6 0792590615
Both ulcerative s rollover and Crohn’s disease patients showed
decreased mean CRP levels through day 155 and then leveled off. Treatment-naive
ulcerative colitis ts had a lower mean CRP level at baseline than ulcerative
colitis er patients (228 v. 7.09). Mean CRP levels of the treatment-naive
ulcerative colitis patients remained relatively constant at all time points assessed.
Other Safety Results
No systematic opportunistic infections ding PML) were reported
during the study. One patient tested positive for JC viremia at a single time point,
though was negative for JCV at all other time points. Three of 72 patients (4%) had
positive HAHA results (two of these were transiently positive). The study showed
no evidence of liver toxicity, lymphocytosis, or lymphopenia, or any other drug-
associated laboratory changes.
Conclusions
Vedolizumab administered at 2.0 or 6.0 mg/kg once every 8 weeks for up to
78 weeks achieved target receptor saturations, was associated with durable mean
decreases in disease activity and improved lBDQ scores, was generally safe and well
tolerated, and demonstrated acceptable immunogenicity.
Example 8: Induction of Response and ion in Patients with Moderate
to Severely Active Crohn’s Disease
A randomized, double blind, placebo controlled multi—center study was
completed to te the ion effect of vedolizumab at 300 mg doses
(reconstituted from a formulation of 60 mg/ml antibody in 50 mM histidine, 125
mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3 which was lyophilized),
in TNFa antagonist failure patients at week 6 (after 2 doses—~0 and 2 weeks) and at
week 10 (after 3 doses). The study ted of 416 patients, 75% of whom were
TNFa antagonist failures, and 25% of whom were TNFa e. Demographics and
concomitant IBD medication were balanced across treatment . Baseline
disease characteristics were also balanced across ent groups, except for
baseline disease activity.
DM‘US ”48987760795906! 5
The primary endpoint designated for the study was week 6 remission (%)in
anti-TNF-a antagonist failure population. The key secondary endpoints that were
evaluated (sequential testing ure) were: week 6 remission (%) in overall
population, week 10 remission (%) in anti-TNF-a antagonist failure and overall
population (using Hochberg ure), week 6 and 10 sustained remission (%) in
anti-TNF-a antagonist failure and overall population (using Hochberg procedure),
and week 6 enhanced response (%) in anti—TNF-01 antagonist failure population.
Table 28: Baseline CDAI:
Placebo Vedolizumab -value
TNF ITT: Mean 3061 (554313161 (52.63) 0_._0945
I (Std Dev)Overall ITT: Mean 301.3 ) 1753.3139
Table 29: Induction Study Results: Primary and Key Secondary Endpoints
Endpoints TNF lTT ) Overall lTT (N=416)
PLA voz Diff P- PLA VDZ Diff P-value
N=157 V=158 (RR) value N=207 N=209 (RMF
Primary 12.1 15.2 3.0 % 0.4332
Wk6 % % (1.2)
Remission
121 T6.9 ‘
1st 19.1 0.0478
Secondary % % %
Wk6 (1.6)
2nd 512.1 26.6 14.4 0.001? 13% 428.7 115.5 '<0.0001
Secondary % % % % %
WklO (2.2) (2.2)
Remission
ned L8.3% 12.0 3.7% 0.275512% L15.3 7% 300249
DMiUS 33489877-60792590615
Remission
(both Wk
6& 1 0)
Enhanced 22.3
Response %
(CDAI 1 00)
Table 30: Results in AntieTNF—a Antagonist Na'l've Patients (n=101, 24% of
overall)
) I Placebo % ’j/edolizumab % Difference % 95% Cl
lRemission Week ission Week 6 12 31.4 19.1 (3.3, 35.0)
16 I 35.3 19.2 (2.4, 35.8)
Table 31: Study Results: Clinical Remission at Weeks 6 and 10, Key
Subgroupw—Previous Tx Failures, ITT Overall
Subgroup Variable Placebo VDZ rDiff 95% C1
Any prior anti- N 156 155
LWk6 Rem L
TNF failure (75% 12.8 14.8 2 (-57, 9.7)
of ITT) (%)
WklO Rem 12.8 26.5 13.6 3)
. ‘L
Prior N 45 44
1 + i
immunomodulator Wk 6 Rem 11.1 31.8 20.7 (—0.5,
failure but not (%) 39.7)
anti-TNF failure ‘
Wk10 Rem 15.6 “31.8 16.3 ,
(21% ITT) (%) 33.6)
Prior N 5 9
corticosteroid “1
Wk6 Rem 0 33.3 33.3 (-23.9,
failure only (3% (%) 75.7)
1 ‘
ITT) WklO Rem 0 44.4 ‘444 (-134,
(%) 85.3)
DM_US 33489877-6 079259 0615
The study showed that TNF-a antagonist failure patients required 3 doses for
induction of remission. Remission rates in TNF-a antagonist failure patients
increased between week 6 and week 10, but only for the vedolizumab group (not
placebo). Remission rates for TNF-a antagonist nai've ts did not increase
substantially between week 6 and 10. Of the TNF-a antagonist failure tion
with a high degree of disease severity, 43% never responded to a TNF—a antagonist,
and 45% lost response.
Example 9: Induction and Maintenance of Response and ion in
Patients with Moderately to Severely Active Ulcerative Colitis
A single trial comprising two randomized, double blind, multi-center studies
designed to evaluate induction and maintenance of response and remission in
patients with moderately to severely active ulcerative colitis. aphic and
baseline disease characteristics were able across all treatment groups.
The induction study, using intravenous administration, compared placebo
against vedolizumab, at a 300 mg dose reconstituted from a lyophilized formulation
of 60 mg/ml antibody in 50 mM ine, 125 mM arginine, 0.06% polysorbate 80,
% sucrose, at pH 6.3, with an endpoint at 6 weeks after 2 doses of vedolizumab.
The maintenance study, using the same formulation and route of
administration as the induction study, compared placebo against vedolizumab dosed
every four weeks, and placebo against vedolizumab dosed every eight weeks. Each
patient was age 18—80, sed with moderately to severely active ulcerative
colitis; trated, over the previous 5 year period, an inadequate response to,
loss of response to, or intolerance of at least one conventional therapy (e.g.
corticosteroids); and may be receiving a therapeutic dose of tional therapies
for IBD. The endpoint of this study was at 52 weeks, analyzing the ion
responder population. Both phases of the trial met their primary endpoints, namely,
clinical response in induction and clinical remission in maintenance.
Blood samples were collected to measure concentrations of vedolizumab
during the study. The mean serum concentration of vedolizumab at the end of the
ion phase was 20 to 30 ug/mL. The mean vedolizimab trough serum
DM_US 33489877-60792590615
concentrations at steady state after 30 min IV infusion of 300mg dose administration
were between 9 to 13 pg/mL for the q8wks n and between 35 to 40 pg/mL
for the q4wks regimen. At the end of infusion, the vedolizimab median plasma
concentrations were between 98 and 101 ug/mL for the q8ks (8 weeks) regimen and
around 129 and 137 pg/mL for the q4 wks (4 weeks).
Summaries of the responses of the induction and maintenance studies are
provided in Tables 32-35. A significantly greater proportion of vedolizumab-treated
patients ed clinical response, remission, and mucosal healing at 6 weeks,
compared with placebo (Table 32). 39% of the induction phase intent-to-treat
population had prior anti-TNFa failure. Clinical response and remission rates were
higher in vedolizumab than placebo patients among both those with prior anti-TNF
failure and those with no prior anti-TNF exposure. In preliminary es through
week 6, rates of adverse events (AEs), serious ABS, and e events leading to
study tinuation were higher in the o group than vedolizumab group. A
significantly greater tion of vedolizumab patients than o patients
achieved clinical remission, mucosal healing, and corticosteroid—free remission at 52
wks and durable response and remission (Table 33). 32% of the maintenance study
populationhad prior anti—TNFa failure. Clinical remission and durable clinical
response rates were greater with zumab than placebo in both TNF failure and
TNF naive patients. In the safety population (N=895) for wks 0—52, rates of adverse
events (AEs), serious ABS, and serious infections were similar between vedolizumab
and o groups. No increase in rates of opportunistic or enteric infections was
observed in the vedolizumab group.
Table 32: Induction Study Results—Primary and Key Secondary nts
Efficacy Placebo Vedolizumab Difference/RR P value
Endpoints
fi25.5% 4 L
Clinical 47.1% 21.7%/l.8 <0.0001
Response (%)
Clinical .34% 16.9% ll.5%/3.l 0.0010
Remission (%)
DM~US 33489877-6079259 0615
Mucosal 24.8% 40.9 0.0013
Healing (%) TIGI‘Vo/lo
Table 33: Maintenance Study Results——Primary and Key Secondary
Endpoints
Efficacy Endpoint Placebo TIDZ Q8 VDZ Q4 Differencm
N=126 N=122 N=125 Q8 vs. Pb
Q4 vs. Pb
’ i 3
al ion 15.9 41.8 44.8 26.1/2.7 <0.0001
(%) 29.1/2.8 <0.0001
Durable Response F23.8 56.6 52.0 1 32.8/24 L<0.0001
(%) 28.5/22 <0.0001
Mucosal Healing 19.8 51.6 56.0 32.0/2.6 W
(%) 36.3/2.8 <0.0001
e Remission 8.7 20.5 24.0 11.8/2.4 0.0090
(%) 15.3/2.8 0.0011
Corticosteroid-free 13.9 31.4 45.2 17.6/2.3 0.0133
ion (%) n=72 n=70 N=73 31.4/3.3 <0.0001
Table 34: Induction Study: Clinical Response and ion at 6 Weeks in
Patients with Prior Anti-TNF-01 Antagonist Failure and Without Anti-TNF Exposure,
ITT Population
Patients with Prior Anti-TNF—a Antagonist Failure (39%)
Endpoint Placebo Vedolizumab Difference 95% Cl
N=63 N=82
Clinical W6 39.0 18.4 3.9, 32.9
Response (%)
m “”3
9.8 73‘ -9.8, 22.8
ion (%)
Patients Withoui NF-(x Antagonist Exposure‘(55%)
H 1
Placebo Vedolizumab Difference 95% Cl
DMWUS 33489877-6 0792590615
al 13.7, 39.9
Response (“/0
Clinical 2.4, 30.2
Remission (%)
Table 35: al Remission and Durable Clinical Response at 52 Weeks:
Patients with Prior Anti-TNF-a Antagonist Failure or Without Anti—TNF-a
Antagonist re ITT Population
Patients with Prior Anti-TNF—a Antagonist Failure (32%) ’1
Endpoint o VDZ TVDZ Difference 95% CT1
N=38 Q8Wks Q4Wks Q8wks vs
N=43 N=40 Placebo
Q4 Wks vs.
Placebo
Clinical remission 5.3 37.2 35.0 31.9 W
(%) 29.7 51.4
7.4,
49.4
Durable Clinical 15.8 46.5 42.5 30.7 11.8,
Response (%) 26.7 49.6
7.5,
45.9
H A
Patients without Anti-TNF—u Antagonist Expglosure (60%)
‘ 7
Placebo VDZ VDZ Fiference 95% Cl
N:79 Q8wks Q4wks Q8wks vs.
N=72 N=73 Placebo
Q4wks vs.
Placebo
' ’ ' 7
Clinical Remission 19.0 45.8 47.9 26.8 12.4,
(%) 29.0 41.2
DM_US 33489877—6 0792590615
14.6,
43.3
Durable Clinical 56.2 38.7 24.0,
Response (°/o) 29.6 53.4
14.6,
44.6
Example 10: Induction and Maintenance of Response and Remission in
Patients with Moderately to Severely Active Crohn’s Disease
A single trial comprising two randomized, double blind, multi—center studies
designed to te induction and maintenance of response and remission in
patients with moderately to severely active Crohn’s Disease. aphic and
ne disease teristics were comparable across all treatment groups.
The induction study, using intravenous administration, compared placebo
against vedolizumab, at a 300 mg dose reconstituted from a lyophilized formulation
of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80,
% e, at pH 6.3, with an endpoint at 6 weeks after 2 doses of vedolizumab.
The maintenance study, using the same formulation and route of
administration as the induction study, compared placebo against zumab dosed
every four weeks, and o against vedolizumab dosed every eight weeks. The
endpoint of this study was at 52 weeks, analyzing the induction responder
population.
singly, this study showed that Q4 and Q8 week groups yielded very
similar results. Summaries of the responses of the induction and maintenance
studies are provided in Tables 36—39. A cantly greater proportion of
vedolizumab-treated patients achieved clinical remission and enhanced response,
compared with placebo (Table 36). Clinical remission and enhanced response rates
were higher in vedolizumab than placebo patients among both those with prior anti~
TNF failure and those with no prior anti-TNF exposure. Rates of adverse events
(AE5), serious ABS, and serious infections were similar between vedolizumab
and placebo groups. No increase in rates of unistic or enteric infections was
observed in the vedolizumab group.
DMAUS 77~6 0792590615
Table 36: Induction Study Results——Primary and Secondary Endpoints
Endpoints Placebo zumab Adjusted anlue
N=148 N=220 Difference/RR
Clinical 7‘
6.8% “774.5% 7.8%/2.1 ”777070206
Remission (%)
L i
Enhanced 725.7% 731.4% 5.7%/1.2 0.2322
Response (%)
Wan CRP “717—36 ‘
—2.9 “7.9288 __‘
Change N=147 N=220
(Hg/mL)
Table 37: Maintenance Study Results—Primary and Key Secondary
Endpoints
Efficacy Endpoint Placebo VDZ Q8 VDZ Q4 Adj. P
N=153 N=154 N=154 Difference/RR value
Q8 vs. Pb
Q4 vs. Pb
Clinical ion 21.6 39.0 36.4 17.4/1.8 00007-1
L(%) .7 0.0042
Enhanced Response 30.1 43.5 45.5 13.4/1.4 0.0132
(%) 15.3/1.5 0.0053
i A
Corticosteroid—free 15.9 31.7 28.8 15.9/2.0 70.0154
Remission (%) N=82 N=82 N=80 8 0.0450
V 4
e Remission 14.4 21.4 16.2 7.2/1.5 71-01036 J
(%) 2.0/1.1 0.6413
Table 38: Clinical Remission and Enhanced Response at 6 Weeks in Patients
with Prior NF-a Antagonist Failure and Without Anti—TNF Exposure, ITT
Population
DMAUS 334898774: 079259 0615
Patients with Prior Anti—TNF-Ol Antagonist Failure (48%)
nt Placebo “"Vedclizumab j “
Difference 95% Cl
N=70 N=105
r ”‘— ”l
Cllmcal. . 4.3 10.5 6.2 (-91, 21.3)
Remission (%)
R J
Enhanced 22.9 F238 1.0 (—11.8, 13.7)
Response (%)
1 _.1
Patients Without Anti-TNF-a Antagonist re (50%)
~ 4
Placebo Vedolizumab ence 95% Cl
N=76 N=130109
__J J
Clinical 9.2 17.4 8.2 (—1.4, 17.9)
Remission (%)
‘ '
Enhanced 30.3 42.2 I 11.9 r(19, 25.8) 1
Response (%)
Table 39: Clinical Remission and Enhanced Response at 52 Weeks: Patients
with Prior Anti-TNF—a Antagonist Failure or Without Anti-TNF-a Antagonist
Exposure ITT Population
Patients with Prior Anti-TNFea Antagonist Failure (51%)
Endpoint Placebo VVDZ VDZ Difference 95% Cl
N=78 Q8Wks Q4Wks Q8wks vs
N=82 N=77 Placebo
Q4 wks vs.
L 1
al remission“ 12.8 38.0 T
27.3 15.2 (3.0,
(%) 14.5 27.5)
(2.0,
26.9)
‘ '
Enhanced 76.5 29.3 37.7 W88 “(4.6,
Response (%) 17.1 22.1)
(3.1,
DM_US 33489877~60792590615
31.2)
L- J
aAntagonist Exposure (45%)
*Piaeebo voz VDZ Difference 95% Cl
N=71 Q8wks Q4Wks Q8wks vs.
N=66 N=7I Placebo
Q4Wks vs.
Placebo
Clinical
26.8 551.1 46.5 524.8 (8.9, ~l
Remission (%) 19.7 40.6)
(4.2,
.2)
Enhanced 38.0 60.6 53.5 22.6 (6.3,
Response (%) 15.5 38.9)
(-0.7,
31.7)
Table 40. Summary of Sequences
SEQ ID NO: uence Shown Description 1
1 DNA encoding heavy
chain of humanized anti—
a4B7 immunoglobulin
2 Amino acid sequence of
heavy chain of humanized
anti—(1407
immunoglobulin
L3 %
DNA encoding the light
chain of humanized anti-
a4B7 immunoglobulin
DMHUS 84898776079259.0615
4 Amino acid sequence of
light chain of humanized
anti-(1467
immunoglobulin
b Mature humanized light
chain of LDP-02
6 I
Generic human kappa
light chain constant region
7 RF1G. 4 Generic murine kappa
light chain constant region
8 kReferenced on page 30 CDRl of heavy chain
mouse ACT-1 antibody
SYWMH
9 fReferenced on page 30 CDR2 of heavy chain
mouse ACT-l antibody
EIDPSESNTNYNQKFKG
Referenced on page 30 bC-IDR3 of heavy chain
mouse ACT—1 antibody
GGYDGWDYAIDY
11 Referenced on page 30 CDRl of light chain
mouse ACT-l antibody
RSSQSLAKSYGNTYLS
4 A
12 Referenced on page 30 CDR2 of light chain
mouse ACT—1 antibody
Fl3 V
nced on page 30 TDR3 of light chain
LQGTHQPYT mouse ACT-l antibody
#14 rhuman GM§O7 CL
antibody kappa light chain
L variable region
DM_US 77-6 0792590615
Human 21/28 CL antibody
heavy chain variable
region
While this invention has been particularly shown and described with references to
preferred embodiments thereof, it will be understood by those skilled in the art that various
changes in form and details may be made therein without ing from the scope of the
invention encompassed by the appended claims.
The reference in this ication to any prior publication (or information derived
from it), or to any matter which is known, is not, and should not be taken as an
acknowledgment or ion or any form of suggestion that that prior publication (or
ation derived from it) or known matter forms part of the common general knowledge in
the field of endeavour to which this specification relates.
Throughout this specification and the claims which follow, unless the context requires
otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be
understood to imply the inclusion of a stated integer or step or group of integers or steps but
not the exclusion of any other integer or step or group of integers or steps.
Claims (9)
1. A stable pharmaceutical formulation comprising a mixture of a non-reducing sugar, an anti-α4β7 antibody and one free amino acid, wherein the ation is lyophilized, and the molar ratio of non-reducing sugar to 4β7 antibody (mole:mole) is 700:1, 5 wherein the free amino acid to antibody molar ratio is at least 250:1, and wherein the antibody comprises a heavy chain variable region comprising a complementarity determining region (CDR1) as set forth in SEQ ID NO:8, a CDR2 as set forth in SEQ ID NO:9, and a CDR3 as set forth in SEQ ID NO:10, and comprises a light chain variable region comprising a CDR1 as set forth in SEQ ID NO:11, a CDR2 as set forth 10 in SEQ ID NO:12, and a CDR3 as set forth in SEQ ID NO:13.
2. The pharmaceutical formulation of claim 1, wherein said formulation further comprises a buffering agent.
3. The pharmaceutical formulation of claim 1, wherein said non-reducing sugar is ed from the group consisting of mannitol, sorbitol, sucrose, trehalose and 15 combinations thereof.
4. The pharmaceutical formulation according to claim 3, n said non-reducing sugar is e.
5. The pharmaceutical formulation of claim 4, wherein the sucrose amount is 45% to 55% sucrose (w/w). 20
6. The pharmaceutical formulation according to any of the previous claims, wherein said ation comprises more than 25% of anti-α4β7 dy by weight.
7. The pharmaceutical formulation of claim 2, wherein said buffering agent is histidine.
8. The pharmaceutical formulation of claim 7, wherein the histidine amount is 3% to 6% (w/w). 25
9. The pharmaceutical formulation of claim 1, wherein said ation further comprises a surfactant.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161481533P | 2011-05-02 | 2011-05-02 | |
US61/481,533 | 2011-05-02 | ||
US201161550545P | 2011-10-24 | 2011-10-24 | |
US61/550,545 | 2011-10-24 | ||
US201261585859P | 2012-01-12 | 2012-01-12 | |
US61/585,859 | 2012-01-12 | ||
PCT/US2012/036072 WO2012151248A2 (en) | 2011-05-02 | 2012-05-02 | FORMULATION FOR ANTI-α4β7 ANTIBODY |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ617833A NZ617833A (en) | 2016-04-29 |
NZ617833B2 true NZ617833B2 (en) | 2016-08-02 |
Family
ID=
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