U.S. patent application number 15/927032 was filed with the patent office on 2018-07-26 for methods of treating ulcerative colitis.
The applicant listed for this patent is Millennium Pharmaceuticals, Inc.. Invention is credited to Irving H. Fox, Catherine Schloz.
Application Number | 20180207279 15/927032 |
Document ID | / |
Family ID | 46085207 |
Filed Date | 2018-07-26 |
United States Patent
Application |
20180207279 |
Kind Code |
A1 |
Fox; Irving H. ; et
al. |
July 26, 2018 |
METHODS OF TREATING ULCERATIVE COLITIS
Abstract
Methods for maintaining clinical remission of ulcerative colitis
in a human patient are described comprising administration of an
antibody that has binding specificity for human alpha4beta7
integrin using a safe dosing regimen of these antibody formulations
that is easy to follow, and which results in a therapeutically
effective amount of the anti-alpha4beta7 antibody in vivo.
Inventors: |
Fox; Irving H.; (Wellesley,
MA) ; Schloz; Catherine; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Millennium Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
46085207 |
Appl. No.: |
15/927032 |
Filed: |
March 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15214993 |
Jul 20, 2016 |
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15927032 |
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13462414 |
May 2, 2012 |
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15214993 |
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61585859 |
Jan 12, 2012 |
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61550545 |
Oct 24, 2011 |
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61481533 |
May 2, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
A61P 1/04 20180101; A61P 1/00 20180101; C07K 16/2839 20130101; A61K
9/19 20130101; A61K 2039/505 20130101; C07K 2317/94 20130101; A61K
47/183 20130101; A61K 2039/54 20130101; A61K 47/26 20130101; C07K
2317/565 20130101; C07K 2317/92 20130101; A61K 39/39591 20130101;
C07K 2317/14 20130101; A61K 9/0019 20130101; A61P 29/00 20180101;
A61P 37/00 20180101; A61P 37/02 20180101; A61K 2039/545
20130101 |
International
Class: |
A61K 47/26 20060101
A61K047/26; A61K 9/19 20060101 A61K009/19; A61K 39/395 20060101
A61K039/395; A61K 47/18 20170101 A61K047/18; A61K 9/00 20060101
A61K009/00; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method for treating a human patient suffering from ulcerative
colitis, wherein the method comprises the steps of: administering
to a patient suffering from ulcerative colitis, an initial 300 mg
dose of a humanized antibody having binding specificity for human
alpha4beta7 integrin, administering a second subsequent dose of 300
mg of the humanized antibody two weeks after the initial dose;
administering a third subsequent dose of 300 mg of the humanized
antibody six weeks after the initial dose; and administering
subsequent doses of 300 mg of the humanized antibody every four
weeks or every eight weeks after the third subsequent dose of the
humanized antibody as needed; wherein the humanized antibody
comprises light chain CDRs as set forth in SEQ ID NO: 11 (CDR1),
SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3), and comprises heavy
chain CDRs as set forth in SEQ ID NO: 8 (CDR1), SEQ ID NO: 9
(CDR2), and SEQ ID NO: 10 (CDR3).
2. The method of claim 1, wherein the patient had a lack of an
adequate response with, loss response to, or was intolerant to
treatment with at least one of an immunomodulator, a tumor necrosis
factor-alpha antagonist, or combinations thereof.
3. The method of claim 1, which induces a clinical response or
clinical remission of ulcerative colitis in the human patient.
4. The method of claim 1, where the patient previously received
treatment with at least one corticosteroid for ulcerative
colitis.
5. The method of claim 1, wherein the ulcerative colitis is
moderate to severely active ulcerative colitis.
6. The method of claim 1, wherein the dosing regimen results in a
reduction, elimination or reduction and elimination of
corticosteroids use by the patient.
7. A method for treating a human patient suffering from ulcerative
colitis, wherein the method comprises the steps of: administering
to a patient suffering from ulcerative colitis, an initial 300 mg
dose of vedolizumab, administering a second subsequent dose of 300
mg of vedolizumab two weeks after the initial dose; administering a
third subsequent dose of 300 mg of vedolizumab six weeks after the
initial dose; and administering subsequent doses of 300 mg of
vedolizumab every four weeks or every eight weeks after the third
subsequent dose.
8. The method of claim 7, wherein the patient had a lack of an
adequate response with, loss response to, or was intolerant to
treatment with at least one of an immunomodulator, a tumor necrosis
factor-alpha antagonist, or combinations thereof.
9. The method of claim 7, which induces a clinical response or
clinical remission of ulcerative colitis in the human patient.
10. The method of claim 7, where the patient previously received
treatment with at least one corticosteroid for ulcerative
colitis.
11. The method of claim 7, wherein the ulcerative colitis is
moderate to severely active ulcerative colitis.
12. The method of claim 7, wherein the dosing regimen results in a
reduction, elimination or reduction and elimination of
corticosteroids use by the patient.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Non-Provisional
application Ser. No. 13/462,414 filed on May 2, 2012, which claims
the benefit of U.S. Provisional Application 61/585,859 filed on
Jan. 12, 2012, U.S. Provisional Application 61/550,545 filed on
Oct. 24, 2011, and U.S. Provisional Application 61/481,533 filed on
May 2, 2011. The entire contents of the foregoing applications are
hereby incorporated by reference.
SEQUENCE LISTING
[0002] 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 Apr. 30, 2012, is named 92596614.txt and is 17,047 bytes in
size.
BACKGROUND OF THE INVENTION
[0003] Advances in biotechnology have made it possible to produce a
variety of proteins for pharmaceutical applications using
recombinant DNA techniques. Because proteins are larger and more
complex than traditional organic and inorganic drugs (i.e.
possessing multiple functional groups in addition to complex
three-dimensional structures), the formulation of such proteins
poses special problems. For a protein to remain biologically
active, a formulation must preserve the conformational integrity of
at least a core sequence of the protein's amino acids, while at the
same time protecting the protein's 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 al., J. Pharm Sci. 96:1-26
(2007)). 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 reported (See e.g., Wang et
al.).
[0004] 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 complicates the
formulation of such systems. Moreover, the excipients included in
antibody formulations preferably minimize any potential immune
response.
[0005] In the case of antibodies, preservation of the
conformational integrity is even more important. Degradation
pathways for proteins can involve chemical instability (i.e., any
process which involves 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.
Physical instability can result from denaturation, aggregation,
precipitation or adsorption, for example. The four most common
protein degradation pathways are protein fragmentation,
aggregation, deamidation, and oxidation. Consequences of chemical
or physical instability of therapeutic protein include a 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.
[0006] Freeze-drying is a commonly employed technique for
preserving proteins; freeze-drying serves to remove water from the
protein preparation of interest. Freeze-drying, or lyophilization,
is a process by which the material to be dried is first frozen and
then the ice or frozen solvent is removed by sublimation 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 protein
formulation (Pikal M., Biopharm. 3(9)26-30 (1990) and Arakawa et
al. Pharm. Res. 8(3):285-291 (1991)).
[0007] 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 associated
with leukocyte recruitment to the gastrointestinal tract as a
result of leukocyte binding to cells expressing MAdCAM. U.S.
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
.alpha.4.beta.7 integrin. U.S. 2005/0095238 further describes
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-.alpha.4.beta.7 antibody or the specific
doses and dose regimens described and claimed herein. Importantly,
the aforementioned patents do not disclose formulations, doses, and
dose regimens that provide for the methods of treatment (supported
by clinical trial data) described and claimed herein.
[0008] 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 compounds, and to
develop formulations, preferably intravenous formulations, 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
[0009] The invention relates to the identification of a
non-reducing sugar and at least one amino acid, as useful
excipients for formulating anti-.alpha.4.beta.7 antibody
formulations whose instability makes them susceptible to
deamidation, oxidation, isomerization and/or aggregation. The
formulation improves stability, reduces aggregate formation and
retards degradation of the antibody therein.
[0010] Thus, in a first aspect, the invention relates to a stable
formulation comprising a mixture of a non-reducing sugar, an
anti-.alpha.4.beta.7 antibody and at least one free amino acid, and
the molar ratio of non-reducing sugar to anti-.alpha.4.beta.7
antibody (mole:mole) is greater than 600:1. The formulation may be
a liquid formulation or a dry formulation (e.g., lyophilized). The
formulation can also contain a buffering agent. In some
embodiments, the non-reducing sugar is mannitol, sorbitol, sucrose,
trehalose, or any combination thereof.
[0011] In some embodiments, the free amino acid of the formulation
is histidine, alanine, arginine, glycine, glutamic acid, or any
combination thereof. The formulation can comprise between about 50
mM to about 175 mM of free amino acid. The formulation can comprise
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.
[0012] The formulation can also contain a surfactant. The
surfactant can be polysorbate 20, polysorbate 80, a poloxamer, or
any combination thereof.
[0013] In some aspects, the formulation can minimize immunogenicity
of the anti-.alpha.4.beta.7 antibody.
[0014] The formulation, e.g., in the dried state, can be stable for
at least three months at 40.degree. C., 75% relative humidity
(RH).
[0015] In another aspect, the formulation is lyophilized and
comprises at least about 5% to about 10% anti-.alpha.4.beta.7
antibody before lyophilization. The formulation can contain at
least about 6% anti-.alpha.4.beta.7 antibody before lyophilization.
The formulation can be reconstituted from a lyophilized formulation
(e.g., reconstituted to comprise a stable liquid formulation).
[0016] In another aspect, the invention relates to a stable
formulation comprising a mixture of a non-reducing sugar, an
anti-.alpha.4.beta.7 antibody and at least one free amino acid, and
the molar ratio of non-reducing sugar to anti-.alpha.4.beta.7
antibody (mole:mole) is greater than 600:1 and the ratio of free
amino acid to anti-.alpha.4.beta.7 antibody (mole:mole) is greater
than 250:1.
[0017] In another aspect, the invention relates to a stable liquid
formulation comprising in aqueous solution with a non-reducing
sugar, an anti-.alpha.4.beta.7 antibody and at least one free amino
acid, wherein the molar ratio of non-reducing sugar to
anti-.alpha.4.beta.7 antibody (mole:mole) 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
anti-.alpha.4.beta.7 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.
[0018] In another aspect, the invention relates to a liquid
formulation comprising at least about 60 mg/ml anti-.alpha.4.beta.7
antibody, at least about 10% (w/v) non-reducing sugar, and at least
about 125 mM of one or more free amino acids.
[0019] In another aspect, the invention relates to a liquid
formulation comprising at least about 60 mg/ml anti-.alpha.4.beta.7
antibody, at least about 10% (w/v) non-reducing sugar, and at least
about 175 mM of one or more free amino acids
[0020] In still yet a further aspect, the invention also relates to
a dry, e.g., lyophilized formulation comprising a mixture of a
non-reducing sugar, an anti-.alpha.4.beta.7 antibody, histidine,
arginine, and polysorbate 80, wherein the formulation is in solid
form, and the molar ratio of non-reducing sugar to
anti-.alpha.4.beta.7 antibody (mole:mole) is greater than
600:1.
[0021] In still yet a further aspect, the invention relates to a
lyophilized formulation comprising a mixture of a non-reducing
sugar, an anti-.alpha.4.beta.7 antibody, histidine, arginine, and
polysorbate 80. In this aspect, the molar ratio of non-reducing
sugar to anti-.alpha.4.beta.7 antibody (mole:mole) is greater than
600:1. Furthermore, the molar ratio of arginine to
anti-.alpha.4.beta.7 antibody (mole:mole) in the formulation is
greater than 250:1.
[0022] In another aspect, the invention relates to a method of
making a formulation described herein, comprising maintaining the
product temperature below the collapse temperature during primary
drying. The method can also contain an annealing step.
[0023] 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
specificity for human .alpha.4.beta.7 integrin, wherein the
humanized immunoglobulin or antigen-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-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 thereof 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
inflammatory bowel disease of the patient; and further wherein the
humanized immunoglobulin or antigen-binding fragment has binding
specificity for the .alpha.4.beta.7 complex, wherein the
antigen-binding region comprises three complementarity determining
regions (CDR1, CDR2, and CDR3) of a light chain variable region and
three complementarity determining regions (CDR1, CDR2, and CDR3) of
a heavy chain variable region of the amino acid sequence set forth
below: light chain: CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ
ID NO:11; heavy chain: CDR1 SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3
SEQ ID NO:14.
[0024] 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
specificity for human .alpha.4.beta.7 integrin, wherein the
humanized immunoglobulin or antigen-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-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 thereof 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
inflammatory bowel disease of the patient; and further wherein the
humanized immunoglobulin or antigen-binding fragment has binding
specificity for the .alpha.4.beta.7 complex, wherein the
antigen-binding region comprises three complementarity determining
regions (CDR1, CDR2, and CDR3) of a light chain variable region and
three complementarity determining regions (CDR1, CDR2, and CDR3) of
a heavy chain variable region of the amino acid sequence set forth
below: light chain: CDR1 SEQ ID NO:9, CDR2 SEQ ID NO:10, CDR3 SEQ
ID NO:11; heavy chain: CDR1 SEQ ID NO:12, CDR2 SEQ ID NO:13, CDR3
SEQ ID NO:14.
[0025] In some aspects the method of treatment with the
anti-.alpha.4.beta.7 antibody formulation, the dose, or the dose
regimen can minimize immunogenicity of the anti-.alpha.4.beta.7
antibody.
[0026] The patient may have had a lack of an adequate response
with, loss response to, or was intolerant to treatment with at
least one of an immunomodulator, a tumor necrosis factor-alpha
(TNF-.alpha.) antagonist or combinations thereof.
[0027] The inflammatory bowel disease can be Crohn's disease or
ulcerative colitis. The inflammatory bowel disease can be moderate
to severely active ulcerative colitis.
[0028] The dosing regimen can result in mucosal healing in patients
suffering from moderate to severely active ulcerative colitis.
[0029] The patient may have previously 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.
[0030] In some aspects, the humanized immunoglobulin or
antigen-binding fragment thereof is administered in a final dosage
form at a concentration of between about 1.0 mg/ml to about 1.4
mg/ml. The humanized immunoglobulin or antigen-binding fragment
thereof can be administered in a final dosage form of about 1.2
mg/ml. The humanized immunoglobulin or antigen-binding fragment can
be administered to the patient in about 30 minutes.
[0031] The humanized immunoglobulin or antigen-binding fragment
thereof can be reconstituted from a lyophilized formulation.
[0032] The humanized immunoglobulin or antigen-binding fragment
thereof can be reconstituted to comprise a stable liquid
formulation.
[0033] In some aspects, the dosing regimen does not alter the ratio
of CD4 to CD8 in cerebrospinal fluid of patients receiving said
treatment.
[0034] The patient can be a person 65 years of age or older and
does not require any adjustment of the dosing regimen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1A is an illustration of a nucleotide sequence (SEQ ID
NO:1) encoding the heavy chain of a humanized anti-.alpha.4.beta.7
immunoglobulin, and FIG. 1B is an illustration of the deduced amino
acid sequence of the heavy chain (SEQ ID NO:2). The nucleotide
sequence contains cloning sites (lower case), Kozak sequence (upper
case, nucleotides 18-23 of SEQ ID NO:1) and leader sequence (lower
case, nucleotides 24-86 of SEQ ID NO:1) at the 5' end of the heavy
chain. The open reading frame of the nucleotide sequence is
nucleotides 24-1433 of SEQ ID NO:1.
[0036] FIG. 2 is an illustration of a nucleotide sequence (SEQ ID
NO:3) encoding the light chain of a humanized immunoglobulin
referred to herein as vedolizumab, and the deduced amino acid
sequence (SEQ ID NO: 4) of the light chain. The nucleotide sequence
contains cloning sites (lower case), Kozak sequence (upper case,
nucleotides 18-23 of SEQ ID NO:3) and leader sequence (lower case,
nucleotides 24-80 of SEQ ID NO:3) at the 5' end of the heavy chain.
The open reading frame of the nucleotide sequence is nucleotides
24-737 of SEQ ID NO:3.
[0037] FIG. 3 is an alignment of the amino acid sequences of (A)
the mature humanized light chain (amino acids 20-238 of SEQ ID
NO:4) of the humanized immunoglobulin referred to herein as
vedolizumab and (B) the mature humanized light chain of the
humanized immunoglobulin referred to herein as LDP-02 (SEQ ID
NO:5). (Regarding LDP-02, see, WO 98/06248 and Feagan et al., N.
Eng. J. Med. 352:2499-2507 (2005). Feagan et al. describe a
clinical study of LDP-02, but in the article they refer to LDP-02
as MLN02.) The alignment illustrates that the amino acid sequences
of the light chains of vedolizumab and LDP-02 differ at positions
114 and 115 of the mature light chains.
[0038] FIG. 4 is an alignment of amino acid sequences of (A) a
generic human kappa light chain constant region (SEQ ID NO:6) and
(B) a generic murine kappa light chain constant region (SEQ ID
NO:7). The amino acid residues Thr and Val (which are present at
positions 114 and 115 of the mature vedolizumab light chain (amino
acids 133 and 134 of SEQ ID NO:4)) are present in the constant
region of the human kappa light chain, whereas the amino acid
residues Ala and Asp (which are present at positions 114 and 115 of
the mature LDP-02 light chain (SEQ ID NO:5)) are present in the
constant region of the mouse kappa light chain.
[0039] FIG. 5 is a map of vector pLKTOK38D (also referred to as
pTOK38MLN02-TV), which encodes the humanized heavy chain and the
humanized light chain of MLN02, and is suitable for producing
vedolizumab in CHO cells. (See, U.S. Patent Application Publication
No. 2004/0033561 Al which discloses pLKTOK38. pLKTOK38D is a
variant of pLKTOK38 in which the restriction sites indicated on the
map flank the sequence encoding the light chain variable
region.)
[0040] FIG. 6A shows the predicted models for change in percent
monomer, change in percent aggregate, and change in percent major
isoform of the anti-.alpha.4.beta.7 lyophilized formulation. The
models are based on statistical analysis of the data presented 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. FIG. 6B shows alternative models based on the
statistical analysis of 40.degree. C. data from Tables 1-3 when the
input factors are pH, sugar:protein molar ratio, and
arginine:protein 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.
[0041] FIG. 7A shows the amino acid sequences of the mature human
GM607'CL antibody kappa light chain variable region and FIG. 7B
shows the human 21/28'CL heavy chain variable region.
[0042] FIG. 8 is a graph showing that solids and load affect drying
time (the numbers in the lines represent the number of minutes of
drying time).
[0043] FIG. 9 is a graph showing vedolizumab did not did not delay
onset of clinical symptoms of experimental autoimmune
encephalomyelitis (EAE) as compared to placebo control. Natalizumab
significantly (p<0.05) delayed onset of clinical symptoms of EAE
as compared to placebo control.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The invention relates to formulations comprising
anti-.alpha.4.beta.7 antibodies. The formulations may be mixtures
comprising non-reducing sugar, anti-.alpha.4.beta.7 antibody and
one or more free amino acids, and the molar ratio of the
non-reducing sugar to anti-.alpha.4.beta.7 antibody is greater than
600 mole non-reducing sugar:1 mole anti-.alpha.4.beta.7 antibody.
The formulations may be in a solid or liquid form.
[0045] Definitions
[0046] The term "pharmaceutical formulation" refers to a
preparation that contains an anti-.alpha.4.beta.7 antibody in such
form as to permit the biological activity of the antibody to be
effective, and which contains no additional components which are
unacceptably toxic to a subject to which the formulation would be
administered.
[0047] A "stable" formulation is one in which the antibody therein
substantially retains its physical stability and/or chemical
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 shelf-life of the formulation. Various analytical
techniques for measuring protein stability are available in the art
and are reviewed in Peptide and Protein Drug Delivery, 247-301,
Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)
and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for
example.
[0048] 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.
[0049] An antibody which is "susceptible to deamidation" is one
comprising one or more residue which has been found to be prone to
deamidate.
[0050] An antibody which is "susceptible to oxidation" is an
antibody comprising one or more residue which has been found to be
prone to oxidation.
[0051] An antibody which is "susceptible to aggregation" is one
which has been found to aggregate with other antibody molecule(s),
especially upon freezing, heating, drying, reconstituting and/or
agitation.
[0052] An antibody which is "susceptible to fragmentation" is one
which has been found to be cleaved into two or more fragments, for
example at a hinge region thereof.
[0053] By "reducing deamidation, oxidation, aggregation, or
fragmentation" is intended to mean preventing or decreasing (e.g.,
to 80%, 60%, 50%, 40%, 30%, 20% or 10% of) the amount of
deamidation, aggregation, or fragmentation relative to the
monoclonal antibody formulated at a different pH or in a different
buffer.
[0054] An "aggregate", "SEC aggregate", or "soluble aggregate" is
more than one and less than or equal to ten antibody proteins
and/or fragments associated together through either covalent,
ionic, or hydrophobic interactions to form a larger protein
body.
[0055] 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.
[0056] As used herein, "biological activity" 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.
[0057] The cell surface molecule, ".alpha.4.beta.7 integrin," or
".alpha.4.beta.7," is a heterodimer of an .alpha..sub.4 chain
(CD49D, ITGA4) and a .beta..sub.7 chain (ITGB7). Each chain can
form a heterodimer with an alternative integrin chain, to form
.alpha..sub.4.beta..sub.1 or .alpha..sub.E.beta..sub.7. Human
.alpha..sub.4 and .beta..sub.7 genes (GenBank (National Center for
Biotechnology Information, Bethesda, Md.) RefSeq Accession numbers
NM_000885 and NM_000889, respectively) are expressed by B and T
lymphocytes, particularly memory CD4+ lymphocytes. Typical of many
integrins, .alpha.4.beta.7 can exist in either a resting or
activated state. Ligands for .alpha.4.beta.7 include vascular cell
adhesion molecule (VCAM), fibronectin and mucosal addressin (MAdCAM
(e.g., MAdCAM-1)).
[0058] As used herein, a human immunoglobulin or antigen-binding
fragment thereof that has "binding specificity for the
.alpha.4.beta.7 complex" binds to .alpha.4.beta.7, but not to
.alpha.4.beta.1 or .alpha.EB7.
[0059] As used herein, an "isotonic" formulation has substantially
the same osmotic pressure as human blood. Isotonic formulations
will generally have an osmotic pressure from about 250 to 350 mOsm.
Isotonicity can be measured using a vapor pressure or ice-freezing
type osmometer, for example.
[0060] As used herein, "buffering 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, phosphate, maleate, cacodylate,
2-[N-morpholino]ethanesulfonic acid (MES),
bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris),
N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and other
organic acid buffers. In another aspect, the buffering agent herein
is histidine or citrate.
[0061] 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.
[0062] A "saccharide" herein is a compound that has a general
formula (CH.sub.2O).sub.n and derivatives thereof, including
monosaccharides, disaccharides, trisaccharides, polysaccharides,
sugar alcohols, reducing sugars, nonreducing sugars, 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, melezitose, raffinose, mannotriose, stachyose, maltose,
lactulose, maltulose, glucitol, maltitol, 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.
[0063] A "surfactant" 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 include polysorbate
(polyoxyethylene sorbitan monolaurate, for example, polysorbate 20
and, polysorbate 80); TRITON (t-Octylphenoxypolyethoxyethanol,
nonionic detergent, Union Carbide subsidiary of Dow Chemical Co.,
Midland Mich.); sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; sorbitan monopalmitate; and the
MONAQUAT series (Mona Industries, Inc., Paterson, N.J.); polyethyl
glycol (PEG), polypropylene glycol (PPG), and copolymers of
poloxyethylene and poloxypropylene glycol (e.g.
Pluronics/Poloxamer, PF68 etc.); etc. In another aspect, the
surfactant is polysorbate 80.
[0064] The term "antibody" herein is used in the broadest sense and
specifically covers full length monoclonal antibodies,
immunoglobulins, polyclonal antibodies, multispecific antibodies
(e.g. bispecific antibodies) formed from at least two full length
antibodies, e.g., each to a different antigen or epitope, and
individual antigen binding fragments, including dAbs, scFv, Fab,
F(ab)'.sub.2, Fab', including human, humanized and antibodies from
non-human species and recombinant antigen binding forms such as
monobodies and diabodies.
[0065] Molar amounts and ratios of anti-.alpha.4.beta.7 antibody to
other excipients described herein are calculated 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 dependent on the cell
line used to express the antibody. Actual antibody molecular weight
can be +/-5% of 150,000 daltons.
[0066] The term "human antibody" includes an antibody that
possesses a sequence that is derived from a human germ-line
immunoglobulin sequence, such as an antibody derived from
transgenic mice having human immunoglobulin genes (e.g., XENOMOUSE
genetically engineered mice (Abgenix, Fremont, Calif.),
HUMAB-MOUSE.RTM., KIRIN TC MOUSE.TM. transchromosome mice,
KMMOUSE.RTM. (MEDAREX, Princeton, N.J.)), human phage display
libraries, human myeloma cells, or human B cells.
[0067] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind 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
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0068] 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 corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). 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.
[0069] "Antigen binding fragments" of the humanized immunoglobulin
prepared in the formulation of the invention comprise at least the
variable regions of the heavy and/or light chains of an
anti-.alpha.4.beta.7 antibody. For example, an antigen binding
fragment of vedolizumab comprises amino acid residues 20-131 of the
humanized light chain sequence of SEQ ID NO:4. Examples of such
antigen binding fragments include Fab fragments, Fab' fragments,
scFv and F(ab').sub.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 cleavage can be used to generate Fab or F(ab').sub.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 upstream of the natural stop
site. For example, a recombinant construct encoding the heavy chain
of an F(ab').sub.2 fragment can be designed to include DNA
sequences encoding the CH.sub.I domain and hinge region of the
heavy chain. In one aspect, antigen binding fragments inhibit
binding of .alpha.4.beta.7 integrin to one or more of its ligands
(e.g. the mucosal addressin MAdCAM (e.g.,MAdCAM-1),
fibronectin).
[0070] 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').sub.2 fragment that has two antigen binding sites
and is still capable of cross-linking antigen.
[0071] "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 terminus 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 residue(s) of the constant domains bear at
least one free thiol group. F(ab').sub.2 antibody fragments
originally were produced as pairs of Fab' fragments which have
hinge cysteines between them. Other chemical couplings of antibody
fragments are also known.
[0072] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. In one aspect, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0073] The term "diabodies" refers to small antibody fragments with
two antigen binding sites, which fragments comprise a variable
heavy domain (V.sub.H) connected to a variable light domain
(V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). 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,
EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993).
[0074] A "full length antibody" is one which comprises an antigen
binding variable region as well as a light chain constant domain
(C.sub.L)and heavy chain constant domains, C.sub.H1, C.sub.H2 and
C.sub.H3. The constant domains may be native sequence constant
domains (e.g. human native sequence constant domains) or amino acid
sequence variants thereof. In one aspect, the full length antibody
has one or more effector functions.
[0075] 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
possess 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, deletions, and/or additions at certain positions
within or adjacent to the amino acid sequence of the main species
antibody, but retain antigen 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.
[0076] "Homology" is defined as the percentage of residues in the
amino acid sequence variant that are identical after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent homology. Methods and computer programs for the
alignment are well known in the art.
[0077] A "therapeutic monoclonal antibody" is an antibody used for
therapy of a human subject. Therapeutic monoclonal antibodies
disclosed herein include anti-.alpha.4.beta.7 antibodies.
[0078] A "glycosylation variant" antibody herein is an antibody
with one or more carbohydrate moeities attached thereto which
differ from one or more carbohydrate moieties attached to a main
species antibody. Examples of glycosylation variants 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.
[0079] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity; 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.
[0080] Depending on the amino acid sequence of the constant domain
of their heavy chains, full length antibodies can be assigned to
different "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., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0081] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0082] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (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 Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII 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 U.S. 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
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in an animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0083] 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
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(See review in M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:33-41 (1995). Other FcRs, including
those 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)).
[0084] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those 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.
[0085] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, 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
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0086] 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 antibody for antigen,
compared to a parent antibody which does not possess those
alteration(s). In one aspect, affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen.
Affinity matured antibodies are produced by procedures known in the
art. Marks et al. Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by:
Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier
et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.
155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9
(1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).
[0087] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. In certain embodiments, the antibody will be purified
(1) to greater than 95% by weight of protein as determined by the
Lowry method, and alternatively, 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 homogeneity 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, isolated antibody will be prepared
by at least one purification step.
[0088] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already 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.
[0089] The antibody which is formulated is substantially pure and
desirably substantially homogeneous (i.e. free from contaminating
proteins etc.). "Substantially pure" antibody means a composition
comprising at least about 90% antibody by weight, based on total
weight of the protein in the composition, at least about 95% or 97%
by weight. "Substantially homogeneous" antibody means a composition
comprising protein wherein at least about 99% by weight of protein
is specific antibody, e.g., anti-.alpha.4.beta.7 antibody, based on
total weight of the protein.
[0090] "Clinical remission" as used herein with reference to
ulcerative colitis subjects refers to a complete 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.
[0091] A "clinical response" as used herein with reference to
ulcerative colitis subjects refers to a reduction 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 Crohn's
disease subjects refers to a 70 point or greater decrease in CDAI
score from baseline (week 0).
[0092] "Mucosal healing" as used herein with reference to
ulcerative colitis subjects refers to an endoscopic subscore of 1
point or less.
[0093] 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 medication required to treat new or unresolved ulcerative
colitis or Crohn's disease symptoms (other than antidiarrheals for
control of chronic diarrhea).
[0094] Formulations
[0095] As described herein, it has been discovered that
anti-.alpha.4.beta.7 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-.alpha.4.beta.7
antibody (mole:mole) is greater than 600:1 are shown herein to be
stable for at least 2 years.
[0096] The present invention provides, in a first aspect, a stable
anti-.alpha.4.beta.7 antibody formulation. In one aspect, the
formulation comprises a buffer, at least one stabilizer and an
anti-.alpha.4.beta.7 antibody. In one aspect, a dry formulation
comprises one or more non-reducing sugars and an
anti-.alpha.4.beta.7 antibody, wherein the ratio of non-reducing
sugar to anti-.alpha.4.beta.7 antibody (mole:mole) 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 fragment thereof, such as a Fab, Fv,
scFv, Fab' or F(ab').sub.2 fragment.
[0097] The formulation can contain any desired non-reducing sugars.
In one aspect, non-reducing sugars that can be included in the
formulation include, for example, mannitol, sorbital, sucrose,
trehalose, raffinose, stachyose, melezitose, dextran, maltitol,
lactitol, isomaltulose, palatinit and combinations thereof. In
another aspect, non-reducing sugars are sucrose, trehalose,
mannitol, and sorbitol. The absolute amount of non-reducing sugar
in the formulation is not critical, but the ratio of non-reducing
sugar to anti-.alpha.4.beta.7 antibody (mole:mole) is greater than
400:1 In another aspect, the ratio of non-reducing sugar to
anti-.alpha.4.beta.7 antibody (mole:mole) is at least about 600:1;
at least about 625:1; at least about 650:1; at least about 675:1,
at least about 700:1; at least about 750:1, at least about 800:1,
at least about 1000:1, at least about 1200:1, at least about
1400:1, at least about 1500:1, at least about 1600:1, at least
about 1700:1, at least about 1800:1, at least about 1900:1, or at
least about 2000:1. Generally, it is desirable that the
non-reducing sugar is present in an amount which reduces soluble
aggregate formation 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-.alpha.4.beta.7 antibody (mole:mole) higher than about 730:1
may give slightly reduced soluble aggregate formation in the
lyophilized state. The sugar:protein 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 mM, about 200 mM to about 350 mM, about 250 mM to about 325 mM,
and about 275 mM to about 300 mM. In another aspect, the amounts of
non-reducing sugar in a dry, (e.g., lyophilized) formulation are in
the range from about 40% to about 70% (w/w of dry formulation). In
another aspect, the amounts of non-reducing sugar in a dry (e.g.,
lyophilized) formulation are in the range 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 1.6:1 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.
[0098] 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 include, for example, histidine, alanine,
arginine, glycine, glutamic acid, serine, lysine, tryptophan,
valine, cysteine and combinations thereof. Some amino acids can
stabilize 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 crystallize when lyophilized as
components of the formulation. Free amino acids, such as glutamic
acid and histidine, alone or in combination, can act as buffering
agents in aqueous solution in the pH range of 5 to 7.5. In still
yet another aspect, the formulation contains histidine, or
histidine and arginine. In still yet a further aspect, the free
amino acid concentrations 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 aspect,
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
embodiments, the amount of histidine in a dry, (e.g., lyophilized)
formulation is greater than about 4% (w/w of the dry formulation)
when the protein amount is about 31% (w/w of the dry formulation)
or greater than about a 0.15:1 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 arginine
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 arginine to protein in the dry formulation. In embodiments
of combinations of amino acids, such as histidine and arginine, the
molar ratio of total amino acid to antibody ratio can be at least
200:1, about 200:1 to about 500:1, or at least 400:1.
[0099] 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
poloxamer (Pluronic.RTM.) 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 pre-dry, (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-.alpha.4.beta.7 antibody can be
present in any desired amount in the formulation, provided that the
ratio of non-reducing sugar to anti-.alpha.4.beta.7 antibody
(mole:mole) is greater than about 600:1. However, the formulation
can contain a high concentration of anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody, about 60 mg/ml
anti-.alpha.4.beta.7 antibody. Dry formulations (e.g., lyophilized)
can contain at least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, or
about 31% or about 32% anti-.alpha.4.beta.7 antibody by weight.
[0100] 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
example, methylamine, ethylenediamine, desferoxamine, trientine,
histidine, malate, phosphonate compounds, e.g., etidronic acid,
ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic
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.
[0101] The formulation can be a liquid or a solid. Liquid
formulations can be aqueous solutions or suspensions, prepared in a
suitable aqueous solvent, such as water or an aqueous/organic
mixture, such as water alcohol mixtures. Liquid formulations 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., 2-8.degree. C.) or frozen (e.g., at -20.degree. C. or
-80.degree. 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 herein, for example by lyophilization,
spray drying, air drying in a film (e.g., for transdermal
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 substantially anhydrous.
Solid formulations 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.,
phosphate-buffered saline, Ringer's (lactated or dextrose)
solution, minimal essential medium, alcohol/aqueous solutions,
dextrose solution, etc. The amount of solvent can result in a
therapeutic protein concentration higher, the same, or lower than
the concentration prior to drying. In one aspect, the reconstituted
anti-.alpha.4.beta.7 antibody concentration is the same
concentration as in the pre-drying liquid formulation.
[0102] 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 liquid, e.g., before drying and/or after reconstitution by
filtration through small pores, through aseptic processing or by
exposure to ultraviolet radiation. Filter pore sizes can be 0.1
.mu.m or 0.2 .mu.m 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-.alpha.4.beta.7 antibody liquid
formulation is sterilized by filtration before drying.
[0103] 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 formulation 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 Techniques for
Biopharmaceutical Development, Rodriguez-Diaz et al. eds. Informa
Healthcare (2005)), including evaluation of aggregate formation
(for example using size exclusion (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 scattering (MALLS)), flow-based microscopic imaging,
electronic impedance (coulter) counting, light obscuration or other
liquid particle counting system, by measuring turbidity, by density
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. Appl. 752:233-245
(2001)), isoelectric focusing (IEF), e.g. capillary technique
(cIEF), or capillary zone electrophoresis; amino-terminal or
carboxy terminal sequence analysis; mass spectrometric analysis;
SDS-PAGE or SEC analysis to compare fragmented, intact and
multimeric (i.e., dimeric, trimeric, etc.) antibody; peptide map
(for example tryptic or LYS-and the like); evaluating biological
activity or antigen binding function of the antibody; and the like.
Biological activity or antigen binding function, e.g., binding of
the anti-.alpha.4.beta.7 antibody to MAdCAM (e.g., MAdCAM-1) or
inhibition of the binding of a cell expressing .alpha.4.beta.7
integrin to MAdCAM (e.g., MAdCAM-1), e.g., immobilized MAdCAM
(e.g., MAdCAM-1), can be evaluated using various techniques
available to the skilled practitioner (see e.g., Soler et al., J.
Pharmacol. Exper. Ther. 330:864-875 (2009)).
[0104] 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 Fourier Transform Infrared Spectroscopy
(FTIR); and measuring thermal transitions in the lyophilized solid
(melting, glass transition, etc.) using Differential Scanning
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 content of a dry
formulation can indicate how likely a formulation will undergo
chemical or physical degradation, with higher moisture leading to
more degradation.
[0105] Stability can be measured at a selected temperature for a
selected time period. In one aspect, a dry, (e.g., lyophilized)
formulation is stable at about 40.degree. 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.degree. C. and/or 25.degree. 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.degree. C. for
at least about 3 months, at least about 6 months, at least about 9
months, at least about 12 months, at least about 18 months, at
least about 24 months, at least about 30 months, at least about 36
months, at least about 42 months, or at least about 48 months.
Furthermore, the liquid formulation may, in some embodiments, be
stable following freezing (to, e.g., -80.degree. C.) and thawing,
such as, for example, following 1, 2 or 3 cycles of freezing and
thawing.
[0106] Instability may involve any one or more of: aggregation
(e.g., non-covalent soluble aggregation (caused by hydrophobic or
charge interactions), 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), isomerization (e.g. Asp isomeriation),
denaturation, clipping/hydrolysis/fragmentation (e.g. hinge region
fragmentation), succinimide formation, N-terminal extension,
C-terminal processing, glycosylation differences, and the like.
[0107] A stable formulation can contribute to a low immunogenicity
of an anti-.alpha.4.beta.7 antibody. An immunogenic
anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody can
have adverse events (e.g., site infusion reaction) upon treatment
or can eliminate anti-.alpha.4.beta.7 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-.alpha.4.beta.7 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 any polysorbate.
[0108] In some embodiments, the formulation can increase the
proportion 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.
[0109] In some embodiments, an anti-.alpha.4.beta.7 antibody
formulation has .gtoreq.50% major charged isoform, .gtoreq.55%
major charged isoform, or 65 to 70% major charged isoform. In other
aspects, a stable anti-.alpha.4.beta.7 antibody formulation has
.ltoreq.45% acidic charged isoforms, .ltoreq.40% acidic charged
isoforms, .ltoreq.30% acidic charged isoforms or 22 to 28% acidic
isoforms. In still other aspects, a stable anti-.alpha.4.beta.7
antibody formulation has .ltoreq.25% basic isoforms, .ltoreq.20%
basic isoforms, .ltoreq.15% basic isoforms, about 5% basic isoforms
or about 10% basic isoforms. In one aspect, a stable
anti-.alpha.4.beta.7 antibody formulation has .gtoreq.55% major
isoform, .ltoreq.30% acidic isoforms and/or .ltoreq.20% basic
isoforms, e.g., as determined by CEX. In another aspect, a stable
anti-.alpha.4.beta.7 antibody formulation has .gtoreq.50% major
isoform, .ltoreq.45% acidic isoforms and/or <10% basic isoforms,
e.g., as determined by cIEF.
[0110] In some aspects, an anti-.alpha.4.beta.7 antibody dry, solid
formulation has .ltoreq.10% moisture content, .ltoreq.5% moisture
content or <2.5% moisture content. The time required for
reconstitution is .ltoreq.60 minutes, .ltoreq.50 minutes or
.ltoreq.40 minutes or .ltoreq.30 minutes or .ltoreq.20 minutes.
[0111] Monomeric content and/or aggregate content (e.g., as dimers,
trimers, tetramers, pentamers, oligomers and higher-order
aggregates), 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 nanoparticle tracking
analysis NTA, NanoSight Ltd, Wiltshire, UK). Resolution,
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.
[0112] In one embodiment, an anti-.alpha.4.beta.7 antibody
formulation has .gtoreq.90% monomeric antibody, .gtoreq.95%
monomeric antibody, or 97 to 99% monomeric antibody. In another
embodiment, the majority of the material in an anti-.alpha.4.beta.7
antibody formulation has an average radius of .ltoreq.20 nm,
.ltoreq.15 nm, .ltoreq.10 nm, or about 5 to about 7 nm. In one
aspect, an anti-.alpha.4.beta.7 antibody formulation has
.gtoreq.80% amount heavy plus light chain by protein analysis. In
one aspect, there is .gtoreq.90% heavy plus light chain. In another
aspect, an anti-.alpha.4.beta.7 antibody formulation has
.ltoreq.10% aggregate, .ltoreq.5% aggregate, .ltoreq.2.5% aggregate
.ltoreq.1.5% aggregate, .ltoreq.1.0% aggregate or .ltoreq.0.5%
aggregate. In another aspect, a stable anti-.alpha.4.beta.7
antibody formulation has .gtoreq.96% monomer and/or .ltoreq.2.5%
aggregate. In yet another aspect, a stable anti-.alpha.4.beta.7
antibody formulation has about 99% monomer and/or about <1%
aggregate.
[0113] Particle sizes, e.g., of aggregates or undissolved
excipient, i.e., in reconstituted formulation can be measured by
light obscuration (e.g., liquid particle counting system (HIAC) by
Hach Ultra Analytics (Grants Pass, Oreg.)), microscopy, coulter
counter, or digital (e.g., flow-based) microscopic imaging based
system such as microfluidics imaging (MFI) by Brightwell (Ottawa,
CA) or FLOWCAM.RTM. Image particle analyzer by Fluid Imaging
Technologies (Yarmouth, Me.). In one aspect, particle size in an
anti-.alpha.4.beta.7 antibody preparation is about 30 .mu.m, about
25 .mu.m, about 10 .mu.m, about 5 .mu.m, about 2 .mu.m or 1 .mu.m
or less. The amount of particles should be minimized in antibody
formulations. In one aspect, the anti-.alpha.4.beta.7 antibody
formulation has less than 6000 particles .gtoreq.10 .mu.m and less
than 600 particles .gtoreq.25 .mu.m diameter in one dose (U.S.
Pharmacopoeia Chp. 788, light obscuration counting method; half
those amounts by microscopic quantification method). In yet another
aspect, an amount of particles per milliliter, e.g., by MFI
measurement, in a dose of an anti-.alpha.4.beta.7 antibody
formulation, e.g., reconstituted formulation is about 500 to about
2000, or about 1000 to about 3000 of 2-10 .mu.m particles per ml,
about 50 to about 350 of .gtoreq.10 .mu.m particles per ml and
about 0 to about 50 of .gtoreq.25 .mu.m particles per ml.
[0114] In one embodiment, an anti-.alpha.4.beta.7 antibody
formulation has a binding affinity of about 60% to about 140% of
the reference standard anti-.alpha.4.beta.7 antibody. In one
aspect, an anti-.alpha.4.beta.7 antibody in a formulation described
herein binds to .alpha.4.beta.7, e.g., on a cell (WO98/06248 or
U.S. Pat. No. 7,147,851), at a value of about 80% to about 120% of
the reference standard. In another embodiment, an
anti-.alpha.4.beta.7 antibody formulation has the ability to
inhibit at least 50% or at least 60% of the binding of a cell
expressing .alpha.4.beta.7 integrin to MAdCAM, e.g., MAdCAM-1, a
MAdCAM-Ig chimera (see U.S. Patent Application Publication No.
20070122404, also for reference standard examples).
[0115] As noted above, freezing of the formulation is specifically
contemplated herein. 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 thawing the
formulation, for example the antibody can be stable after one, two,
three, four, five or more freeze/thaw cycles.
[0116] In some embodiments, the formulation is a liquid formulation
comprising at least about 50 mg/ml to about 100 mg/ml
anti-.alpha.4.beta.7 antibody, a buffering agent (e.g., histidine),
and at least about 9% (w/w) non-reducing sugar (e.g, sucrose,
trehalose or mannitol). In one embodiment, the formulation
comprises at least about 50 to about 80 mg/ml, about 60 mg/ml
anti-.alpha.4.beta.7 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).
[0117] In another embodiment, the formulation comprises at least
about 60 mg/ml anti-.alpha.4.beta.7 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.
[0118] 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-.alpha.4.beta.7 antibody,
histidine, arginine, and polysorbate 80, and the molar ratio of
non-reducing sugar to anti-.alpha.4.beta.7 antibody (mole:mole) is
greater than 600:1.
[0119] In another embodiment, the formulation is a dry, solid,
amorphous formulation (e.g., a lyophilized formulation), comprising
a mixture of a non-reducing sugar, an anti-.alpha.4.beta.7
antibody, histidine, arginine, and polysorbate 80, and the molar
ratio of non-reducing sugar to anti-.alpha.4.beta.7 antibody
(mole:mole) is greater than 600:1.
[0120] In one embodiment, the formulation is a lyophilized
formulation comprising a non-reducing sugar, an
anti-.alpha.4.beta.7 antibody, histidine, arginine and polysorbate
80, and the molar ratio of non-reducing sugar to
anti-.alpha.4.beta.7 antibody (mole:mole) in the formulation is
greater than 600:1.
[0121] In one embodiment, the formulation is a lyophilized
formulation comprising a non-reducing sugar, an
anti-.alpha.4.beta.7 antibody, histidine, arginine and polysorbate
80, wherein the molar ratio of non-reducing sugar to
anti-.alpha.4.beta.7 antibody (mole:mole) in the formulation is
greater than 600:1 and the molar ratio of arginine to
anti-.alpha.4.beta.7 antibody (mole:mole) in the formulation is
greater than 250:1.
[0122] In one embodiment, the formulation is a liquid formulation
and comprises at least about 60 mg/ml anti-.alpha.4.beta.7
antibody, at least about 10% (w/v) non-reducing sugar, and at least
about 125 mM of one or more free amino acids.
[0123] In one embodiment, the formulation is a liquid formulation
and comprises at least about 60 mg/ml anti-.alpha.4.beta.7
antibody, at least about 10% (w/v) non-reducing sugar, and at least
about 175 mM of one or more free amino acids.
[0124] In one embodiment, the formulation is a liquid formulation
and comprises between about 60 mg/ml to about 80 mg/ml
anti-.alpha.4.beta.7 antibody, a buffering agent and at least about
10% (w/w) sugar.
[0125] In one embodiment, the formulation is a liquid formulation
and comprises between about 60 mg/ml to about 80 mg/ml
anti-.alpha.4.beta.7 antibody, histidine and at least about 10%
(w/w) sucrose.
[0126] In one embodiment, the formulation is lyophilized and stored
as a single dose in one vial. The vial is desirably stored at about
2-8.degree. C. until it is administered to a subject in need
thereof. The vial may for example be a 20 or 50 cc vial (for
example 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-.alpha.4.beta.7 antibody. In one aspect,
the vial contains about 300 mg of anti-.alpha.4.beta.7
antibody.
[0127] One or more other pharmaceutically acceptable carriers,
excipients or stabilizers such as those described in Remington: The
Science and Practice of Pharmacy, 21st Edition, Hendrickson, R. Ed.
(2005) may be included in the formulation provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients 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.
[0128] .alpha.4.beta.7 Antibodies
[0129] Anti-.alpha.4.beta.7 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
fragments of any of these types of antibodies, such as Fab, Fv,
scFv, Fab' and F(ab').sub.2 fragments, are also suitable for use in
the formulations.
[0130] The anti-.alpha.4.beta.7 antibody can bind to an epitope on
the .alpha.4 chain (e.g., humanized MAb 21.6 (Bendig et al., U.S.
Pat. No. 5,840,299), on the .beta.7 chain (e.g., FIB504 or a
humanized derivative (e.g., Fong et al., U.S. Pat. No. 7,528,236)),
or to a combinatorial epitope formed by the association of the
.alpha.4 chain with the .beta.7 chain. In one aspect, the antibody
binds a combinatorial epitope on the .alpha.4.beta.7 complex, but
does not bind an epitope on the .alpha.4 chain or the .beta.7 chain
unless the chains are in association with each other. The
association of .alpha.4 integrin with .beta.7 integrin can create a
combinatorial epitope for example, by bringing into proximity
residues present on both chains which together comprise the epitope
or by conformationally exposing on one chain, e.g., the .alpha.4
integrin chain or the .beta.7 integrin chain, an epitopic binding
site that is inaccessible to antibody binding in the absence of the
proper integrin partner or in the absence of integrin activation.
In another aspect, the anti-.alpha.4.beta.7 antibody binds both the
.alpha.4 integrin chain and the .beta.7 integrin chain, and thus,
is specific for the .alpha.4.beta.7 integrin complex. Such
antibodies can bind .alpha.4.beta.7 but not bind .alpha.4.beta.1,
and/or not bind .alpha..sub.E.beta.7, for example. In another
aspect, the anti-.alpha.4.beta.7 antibody binds to the same or
substantially the same epitope as the Act-1 antibody (Lazarovits,
A. I. et al., J. Immunol., 133(4): 1857-1862 (1984), Schweighoffer
et al., J. Immunol., 151(2): 717-729, 1993; Bednarczyk et al., J.
Biol. Chem., 269(11): 8348-8354, 1994). Murine ACT-1 Hybridoma cell
line, which produces the murine Act-1 monoclonal antibody, was
deposited under the provisions of the Budapest Treaty on Aug. 22,
2001, on behalf Millennium Pharmaceuticals, Inc., 40 Landsdowne
Street, Cambridge, Mass. 02139, U.S.A., at the American Type
Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, U.S.A., under Accession No. PTA-3663. In another
aspect, the anti-.alpha.4.beta.7 antibody is a human antibody or an
.alpha.4.beta.7 binding protein using the CDRs provided in U.S.
Patent Application Publication No. 2010/0254975.
[0131] In one aspect, the anti-.alpha.4.beta.7 antibody inhibits
binding of .alpha.4.beta.7 to one or more of its ligands (e.g. the
mucosal addressin, e.g., MAdCAM (e.g., MAdCAM-1), fibronectin,
and/or vascular addressin (VCAM)). Primate MAdCAMs are described in
the PCT publication WO 96/24673, the entire teachings of which are
incorporated herein by this reference. In another aspect, the
anti-.alpha.4.beta.7 antibody inhibits binding of .alpha.4.beta.7
to MAdCAM (e.g., MAdCAM-1) and/or fibronectin without inhibiting
the binding of VCAM.
[0132] In one aspect, the anti-.alpha.4.beta.7 antibodies for use
in the formulations are humanized versions of the mouse Act-1
antibody. Suitable methods for preparing humanized antibodies are
well-known in the art. Generally, the humanized
anti-.alpha.4.beta.7 antibody will contain a heavy chain that
contains the 3 heavy chain complementarity determining regions
(CDRs, CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10)
of the mouse Act-1 antibody and suitable human heavy chain
framework regions; and also contain a light chain that contains the
3 light chain CDRs (CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and
CDR3, SEQ ID NO:13) of the mouse Act-1 antibody and suitable human
light chain framework regions. The humanized Act-1 antibody can
contain any suitable human framework regions, including consensus
framework 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 .kappa. or .lamda. light chains, and/or the .gamma. (e.g.,
.gamma.1, .gamma.2, .gamma.3, .gamma.4), .mu., .alpha. (e.g.,
.alpha.1, .alpha.2), .delta. or .epsilon. heavy chains of human
antibodies, including allelic variants. A particular constant
region (e.g., IgG1), 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 et al., GB 2,209,757 B; Morrison et al., WO
89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994). Humanized
versions of Act-1 antibody were described in PCT publications nos.
WO98/06248 and WO07/61679, the entire teachings of each of which
are incorporated herein by this reference.
[0133] In another aspect, the anti-.alpha.4.beta.7 humanized
antibodies for use in the formulation comprise a heavy chain
variable region comprising amino acids 20 to 140 of SEQ ID NO:2,
and a light chain variable region comprising amino acids 20 to 131
of SEQ ID NO:4 or amino acids 21 to 132 of SEQ ID NO:5. If desired,
a suitable human constant region(s) can be present. For example,
the humanized anti-.alpha.4.beta.7 antibody can comprise a heavy
chain that comprises amino acids 20 to 470 of SEQ ID NO:2 and a
light chain comprising amino acids 21 to 239 of SEQ ID NO:5. In
another example, the humanized anti-.alpha.4.beta.7 antibody can
comprise a heavy chain that comprises amino acids 20 to 470 of SEQ
ID NO:2 and a light chain comprising amino acids 20 to 238 of SEQ
ID NO:4. FIG. 4 shows an alignment which compares the generic light
chains of human antibodies with murine antibodies. The alignment
illustrates that the humanized light chain of vedolizumab (e.g.,
Chemical Abstract Service (CAS, American Chemical Society) Registry
number 943609-66-3), with two mouse residues switched for human
residues, is more human than the light chain of LDP-02 (FIG. 3). In
addition, LDP-02 has the somewhat hydrophobic, flexible alanine 114
and a hydrophilic site (Aspartate 115) that is replaced in
vedolizumab with the slightly hydrophilic hydroxyl-containing
threonine 114 and hydrophobic, potentially inward facing valine 115
residue.
[0134] Further substitutions to the antibody sequence can be, for
example, mutations to the heavy and light chain framework regions,
such as a mutation of isoleucine 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 mutation of alanine to glycine 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 residue 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 (CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and
CDR3, SEQ ID NO:10) of the mouse Act-1 antibody; and replacement of
the light chain CDRs with the light chain CDRs (CDR1, SEQ ID NO:11,
CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1
antibody.
[0135] In some embodiments, the anti-.alpha.4.beta.7 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 NO:2, and a light chain
variable region that has about 95%, 96%, 97%, 98%, or 99% sequence
identity to amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21
to 132 of SEQ ID NO:5. Amino acid sequence identity can be
determined using a suitable sequence alignment algorithm, such as
the Lasergene system (DNASTAR, Inc., Madison, Wis.), using the
default parameters. In an embodiment, the anti-.alpha.4.beta.7
antibody for use in the formulation is vedolizumab (CAS, American
Chemical Society, Registry number 943609-66-3).
[0136] Other .alpha.4.beta.7 antibodies may also be used in the
formulations and dosing regimes described herein. For example, the
.alpha.4.beta.7 antibodies described in US 2010/0254975 (Amgen,
Inc.), incorporated by reference herein in its entirety, are
suitable for use in the formulations and methods of treating
inflammatory bowel disease in an individual.
[0137] The anti-.alpha.4.beta.7 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 sequences,
express an anti-.alpha.4.beta.7 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 sequences; yeast (e.g., Saccharomyces, Pichia) transformed
with recombinant yeast expression vectors containing antibody
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing antibody
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,
3T3, NS0 cells) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K promoter).
For example, mammalian cells such as Chinese hamster ovary cells
(CHO), in conjunction with a vector such as the major intermediate
early gene promoter element from human cytomegalovirus is an
effective expression system for antibodies (Foecking et al., Gene
45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
[0138] 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 desirable. Such vectors 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 & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (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 glutathione-agarose beads followed by elution in
the presence 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.
[0139] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned 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).
[0140] 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 sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo 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 sequences. 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, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0141] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. 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 correct modification and
processing of the foreign protein 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), NS0, HeLa, VERY, baby hamster kidney (BHK), monkey kidney
(COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma cells
(e.g., Hep G2), breast cancer cell lines such as, for example,
BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
[0142] 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-.alpha.4.beta.7
antibody are mammalian cells, such as NS0 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 exogenous gene
confers to the cells which host the construct or vector a selection
advantage to encourage production of the polypeptide encoded by the
exogenous gene. In one embodiment, CHO cells are DG44 cells (Chasin
and Urlaub (1980) PNAS USA 77:4216), comprising the 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., U.S. Pat.
Nos. 5,122,464 or 5,827,739).
[0143] Solid Formulations
[0144] Solid formulations of the invention are generally prepared
by drying a liquid formulation. Any suitable method of drying can
be used, such as lyophilization or spray drying. 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.,
317-367 (1994).) Once the formulation is frozen, the atmospheric
pressure is reduced and the temperature is adjusted to allow
removal of the frozen solvent e.g., through sublimation. This step
of the lyophilization process is sometimes referred to as primary
drying. If desired, the temperature can then be raised to remove
any solvent that is still bound to the dry formulation by
evaporation. This step of the lyophilization process is sometimes
referred to as secondary drying. When the formulation has reached
the desired degree of dryness, the drying process is concluded and
the containers are sealed. The final solid formulation is sometimes
referred to as a "lyophilized formulation" or a "cake." The
lyophilization process can be performed using any suitable
equipment. Suitable lyophilization equipment is available from a
number of commercial sources (e.g., SP Scientific, Stone Ridge,
N.Y.).
[0145] A variety of suitable apparatuses can be used to dry liquid
formulations to produce a solid (e.g., lyophilized) 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 shelves, 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 processes performed using this type of apparatus. Persons of
ordinary skill can easily adapt the parameters described herein to
other types of drying apparatuses if desired.
[0146] 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 -30.degree. C. or less, such as -40.degree. C.
or -50.degree. 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.degree. C., about
20.degree. C., about 30.degree. C., about 40.degree. C. or about
50.degree. C. warmer than the freezing temperature. In one aspect,
the primary drying conditions can be set to maintain the
anti-.alpha.4.beta.7 antibody below the glass transition
temperature or collapse temperature of the formulation. Above the
collapse temperature, the amorphous frozen matrix can 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 formulation 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 measured by
methods known in the art, e.g., by differential scanning
calorimetry (DSC). The collapse temperature can be measured by
methods known in the art, e.g. freeze-drying microscopy. The ratio
of non-reducing sugar to protein (mole:mole) and the amounts of
other formulation components will impact the glass transition
temperature and collapse temperature. In some embodiments, a glass
transition temperature for an .alpha.4.beta.7 antibody formulation
is about -35.degree. C. to about -10.degree. C., about -35.degree.
C. to about -25.degree. C., or about -35.degree. C. to about
-29.degree. C. In another embodiment, the glass transition
temperature of an .alpha.4.beta.7 antibody formulation is about
-29.degree. C. In some embodiments, the glass transition
temperature of an .alpha.4.beta.7 antibody formulation is about
-30.degree. C., about -31.degree. C., about -32.degree. C., about
-33.degree. C., about -34.degree. C., about -35.degree. C. or about
-36.degree. C. In some embodiments, a collapse temperature of an
.alpha.4.beta.7 antibody formulation is about -30.degree. C. to
about 0.degree. C., about -28.degree. C. to about -25.degree. C.,
or about -20.degree. C. to about -10.degree. C. In another
embodiment, the collapse temperature of an .alpha.4.beta.7 antibody
formulation is about -26.degree. C. 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-.alpha.4.beta.7 antibody
formulation.
[0147] 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 of Pharm Sci & Tech, 51: 7-16), coupled with
knowledge of Rp and Kv, allow for understanding the combination and
interaction of input variables including process input variables
such as shelf temperature and pressure and formulation 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 product temperature by the collapse
temperature and equipment capability.
d m dt = A p ( P o - P c ) R p Equation 1 ln P 0 = - 6144.96 / T p
+ 24.0185 Equation 2 dQ dt = A v K v ( T s - T p ) Equation 3 dQ dt
= .DELTA. H s d m dt Equation 4 ##EQU00001##
[0148] Equation 1 relates the sublimation rate (dm/dt) during
primary drying to the internal cross-sectional area of the
container (A.sub.p), the vapor pressure of ice (P.sub.o), the
pressure of the chamber (P.sub.c), and an area normalized mass
transfer resistance for the cake and stopper (R.sub.p). P.sub.o at
the sublimation interface can be determined from Equation 2, where
P.sub.o is related to the temperature of the product ice at the
sublimation interface, which is an approximation from the product
temperature (T.sub.p), which can be measured with a thermocouple at
the bottom of the vial or can be derived from the equations above
when the other variables are determined. Equation 3 relates the
heat transfer rate from the shelf to the vials, where A.sub.v is
the area of the vial, K.sub.v is the heat transfer coefficient of
the vial, T.sub.s is the temperature of the shelf, and T.sub.p is
the product temperature. Equation 4 couples the heat and mass
transfer equations, where .DELTA.H.sub.s is the heat of
sublimation.
[0149] As seen from the equations for primary drying, the shelf
temperature (T.sub.s), the product temperature (T.sub.p), the
chamber pressure (P.sub.c), the mass transfer resistance of the
cake (R.sub.p), and the heat transfer coefficient (K.sub.v) can
affect the sublimation rate.
[0150] 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 lowered again to below
the glass transition temperature of the formulation. Annealing can
be used to crystallize 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 .alpha.4.beta.7 antibody formulation can be at about -30.degree.
C. to about -10.degree. C. or about -25.degree. C. to about
-15.degree. C. In one aspect, an annealing temperature for an
.alpha.4.beta.7 antibody formulation is about -20.degree. C.
[0151] Secondary drying is generally conducted at a temperature
that is above the freezing temperature of the liquid formulation.
For example, secondary drying can be conducted at about 10.degree.
C., about 20.degree. C., about 30.degree. C., about 40.degree. C.
or about 50.degree. C. In one aspect, the temperature for secondary
drying is ambient temperature, e.g., 20-30.degree. C. The time for
secondary drying should be sufficient to reduce the amount of
moisture to <5%.
[0152] In another aspect, the lyophilization cycle includes
freezing at about -45.degree. C., annealing at about -20.degree.
C., refreezing at about -45.degree. C., primary drying at about
-24.degree. C. and 150 mTorr, and secondary drying at about
27.degree. C. and 150 mTorr.
[0153] R.sub.p 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 primary and secondary lyophilization stages can be the
shelf temperature and chamber pressure during each stage of the
drying cycle.
[0154] For scale-up, freeze dryer load and solid content can affect
the drying cycle. Primary drying time can be affected by the solids
content in the formulation. 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 determined, the drying time can
be affected. For example, a high solids content formulation 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 capacity,
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 % increases as the solids content
increases. In one embodiment, the solids content is less than
20-25% and the load is from 25-100%.
[0155] Vial size can be selected 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 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, because larger vials
can be more expensive to store and ship and have a larger headspace
to formulation ratio and may expose a high proportion of the
formulation to the degradative effects of moisture during long term
storage. For a 300 mg dose, anti-.alpha.4.beta.7 antibody
formulation can have a volume of 3 ml, 5 ml, 6 ml, 10 ml, 20 ml, 50
ml or 100 ml prior to lyophilization. In one aspect, the vial size
is 20 ml for a 60 mg/ml solution in a 300 mg dose.
[0156] 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 lyophilized 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 nitrogen or
argon.
[0157] In some embodiments, the pre-lyophilization
anti-.alpha.4.beta.7 antibody formulation volume is the same as the
pre-administration reconstituted solution volume. For example, a
formulation which is about 5.5 ml pre-lyophilization 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-.alpha.4.beta.7 antibody formulation in a
different volume than the reconstituted solution volume. For
example, the anti-.alpha.4.beta.7 antibody formulation can be
lyophilized as a dilute solution, e.g. 0.25.times., 0.5.times., or
0.75.times. and reconstituted to 1.times. 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-.alpha.4.beta.7 antibody formulation can be
reconstituted into a more dilute solution than the pre-lyophilized
formulation.
Treatment with the Antibody Formulation
[0158] In one aspect, the invention provides a method of treating a
disease or disorder in a subject comprising administering to a
subject the anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody
formulation described herein.
[0159] The subject may have had a lack of an adequate response
with, loss of response to, or was intolerant to treatment with an
immunomodulator, a TNF-.alpha. 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 response to corticosteroids refers to signs
and symptoms of persistently active disease despite a history of at
least one 4-week induction regimen that included a dose equivalent
to prednisone 30 mg daily orally for 2 weeks or intravenously 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.
[0160] An immunomodulator may be, for example, oral azathioprine,
6-mercaptopurine, or methotrexate. An inadequate response to an
immunomodulator refers to signs and symptoms of persistently active
disease despite a history of at least one 8 week regimen or oral
azathioprine (.gtoreq.1.5 mg/kg), 6-mercaptopurine (.gtoreq.0.75
mg/kg), or methotrexate (.gtoreq.12.5 mg/week). Intolerance of an
immunomodulator includes, but is not limited to, nausea/vomiting,
abdominal pain, pancreatitis, LFT abnormalities, lymphopenia, TPMT
genetic mutation and/or infection.
[0161] 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 TNF-.alpha. antagonist. A TNF-.alpha. antagonist is,
for example, an agent that inhibits the biological activity of
TNF-.alpha., and preferably binds TNF-.alpha., such as a monoclonal
antibody, e.g., REMICADE (infliximab), HUMIRA (adalimumab), CIMZIA
(certolizumab pegol), SIMPONI (golimumab) or a circulating receptor
fusion protein such as ENBREL (etanercept). An inadequate response
to a TNF-.alpha. 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, follwed 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-.alpha. antagonist refers to recurrence of
symptoms during maintenance dosing following prior clinical
benefit. Intolerance of a TNF-.alpha. antagonist includes, but is
not limited to infusion related reaction, demyelination, congestive
heart failure, and/or infection.
[0162] 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.
[0163] In another aspect, the present invention provides
anti-.alpha.4.beta.7 antibody formulations which (1) can bind
.alpha.4.beta.7 integrin in vitro and/or in vivo; and (2) can
modulate an activity or function of an .alpha.4.beta.7 integrin,
such as (a) binding function (e.g., the ability of .alpha.4.beta.7
integrin to bind to MAdCAM (e.g., MAdCAM-1), fibronectin and/or
VCAM-1) and/or (b) leukocyte infiltration function, including
recruitment and/or accumulation of leukocytes in tissues (e.g., the
ability to inhibit lymphocyte migration to intestinal mucosal
tissue). In one embodiment, an antibody in the formulation can bind
an .alpha.4.beta.7 integrin, and can inhibit binding of the
.alpha.4.beta.7 integrin to one or more of its ligands (e.g.,
MAdCAM (e.g., MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting
leukocyte infiltration of tissues (including recruitment and/or
accumulation of leukocytes in tissues). In another embodiment, an
antibody in the formulation can bind an .alpha.4.beta.7 integrin,
and can selectively inhibit binding of the .alpha.4.beta.7 integrin
to one or more of its ligands (e.g., MAdCAM (e.g., MAdCAM-1),
VCAM-1, fibronectin), thereby inhibiting leukocyte infiltration of
tissues (including recruitment and/or accumulation of leukocytes in
tissues). Such anti-.alpha.4.beta.7 antibody formulations can
inhibit cellular adhesion of cells bearing an .alpha.4.beta.7
integrin to vascular endothelial cells in mucosal tissues,
including gut-associated tissues, lymphoid organs or leukocytes
(especially lymphocytes such as T or B cells) in vitro and/or in
vivo. In yet another embodiment, the anti-.alpha.4.beta.7 antibody
formulation of the present invention can inhibit the interaction of
.alpha.4.beta.7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. In
still yet another embodiment, the anti-.alpha.4.beta.7 antibody
formulation of the present invention can inhibit the interaction of
.alpha.4.beta.7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin
selectively, e.g., without inhibiting the interaction of
.alpha.4.beta.7 with VCAM.
[0164] The anti-.alpha.4.beta.7 antibody formulations of the
present invention can be used to modulate (e.g., inhibit (reduce or
prevent)) binding function and/or leukocyte (e.g., lymphocyte,
monocyte) infiltration function of .alpha.4.beta.7 integrin. For
example, humanized immunoglobulins which inhibit the binding of
.alpha.4.beta.7 integrin to a ligand (i.e., one or more ligands)
can be administered according to the method in the treatment of
diseases associated with leukocyte (e.g., lymphocyte, monocyte)
infiltration of tissues (including recruitment and/or accumulation
of leukocytes in tissues), particularly of tissues which express
the molecule MAdCAM (e.g., MAdCAM-1).
[0165] An effective amount of an anti-.alpha.4.beta.7 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 disease. 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-1) (e.g.,
gut-associated tissues, such as venules of the lamina propria of
the small and large intestine; and mammary gland (e.g., lactating
mammary gland)), can be treated according to the present method.
Similarly, an individual having 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., MAdCAM-1)
can be treated according to the present invention.
[0166] In one embodiment, diseases which can be treated 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 pouchitis resulting after proctocolectomy, and ileoanal
anastomosis. Preferably, the inflammatory bowel disease is Crohn's
disease or ulcerative colitis. 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 ulcerative colitis. Treatment may also result in a
reduction, elimination, or reduction and elimination of
corticosteroid use by the patient.
[0167] Pancreatitis and insulin-dependent diabetes mellitus are
other diseases which can be treated using the formulations of the
invention. It has been reported that MAdCAM (e.g., MAdCAM-1) is
expressed by some vessels in the exocrine pancreas from NOD
(nonobese diabetic) mice, as well as from BALB/c and SJL mice.
Expression of MAdCAM-1 was reportedly induced on endothelium in
inflamed islets of the pancreas of the NOD mouse, and MAdCAM-1 was
the predominant addressin expressed by NOD islet endothelium at
early stages of insulitis (Hanninen, A., et al., J. Clin. Invest.,
92: 2509-2515 (1993)). Treatment of NOD mice with either
anti-MAdCAM (e.g., anti-MAdCAM-1) or anti (37 antibodies prevented
the development of diabetes (Yang et al., Diabetes, 46:1542-1547
(1997)). Further, accumulation of lymphocytes expressing
.alpha.4.beta.7 within islets was observed, and MAdCAM-1 was
implicated in the binding of lymphoma cells via .alpha.4.beta.7 to
vessels from inflamed 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.,
153:1701-1705 (1998)).
[0168] Examples of inflammatory diseases associated with mucosal
tissues which can be treated using a formulation of the invention
include cholecystitis, cholangitis (Adams and Eksteen Nature
Reviews 6:244-251 (2006) Grant et al., Hepatology 33:1065-1072
(2001)), e.g., primary sclerosing cholangitis, Behcet's disease,
e.g., of the intestine, or 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 al. 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., autoimmune gastritis (Katakai
et al., Int. Immunol., 14:167-175 (2002)) and other idiopathic
conditions can be amenable to treatment.
[0169] The invention also relates to a method of inhibiting
leukocyte infiltration of mucosal tissue. The invention also
relates to a method for treating cancer (e.g., an .alpha.4.beta.7
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 mastitis (mammary gland) and
irritable bowel syndrome.
[0170] Diseases or pathogens whose etiologies exploit the
interaction of MAdCAM (e.g., MAdCAM-1) with .alpha.4.beta.7 can be
treated with an anti-.alpha.4.beta.7 antibody in a formulation
described herein. Examples of such diseases include
immunodeficiency disorders, such as caused by human
immunodeficiency virus (see e.g., WO2008140602).
[0171] A formulation of the invention is administered in an
effective amount which inhibits binding of .alpha.4.beta.7 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 .alpha.4.beta.7 integrin-mediated binding and/or signaling,
thereby inhibiting leukocyte adhesion and infiltration and/or
associated cellular responses). An effective amount of an
anti-.alpha.4.beta.7 antibody, e.g., an effective titer sufficient
to maintain saturation, e.g., neutralization, of .alpha.4.beta.7
integrin, can induce clinical response or remission in inflammatory
bowel disease. A formulation of the invention can be administered
in a unit dose or multiple doses. The dosage can be determined by
methods known in the art and can be dependent, for example, upon
the individual's age, sensitivity, tolerance and overall
well-being. Examples of modes of administration include topical
routes such as nasal or inhalational or transdermal administration,
enteral routes, such as through a feeding tube or suppository, 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.
[0172] 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-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody dose can be diluted into a 250 ml
saline or 5% dextrose solution for administration.
[0173] In some aspects, the dosing regimen has two phases, an
induction phase and a maintenance phase. In the induction phase,
the antibody or antigen-binding fragment thereof is administered in
a 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 fragment thereof or for inducing a clinical
response and ameliorating inflammatory bowel disease symptoms. A
patient can be administered an induction phase treatment when first
being treated by an anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody therapy or during
maintenance phase of anti-.alpha.4.beta.7 antibody therapy if there
has been a return of inflammatory bowel disease symptoms, e.g., a
relapse from remission of disease. In some embodiments, the
induction phase regimen results in a higher mean trough serum
concentration, e.g., the concentration just before the next dose,
than the mean steady state trough serum concentration maintained
during the maintenance regimen.
[0174] In the maintenance phase, the antibody or antigen-binding
fragment thereof is administered in a way that continues the
response achieved by induction therapy with a stable level of
antibody or antigen-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-.alpha.4.beta.7 antibody or
antigen-binding fragment thereof, e.g., in a formulation described
herein, by a strategy selected from the group consisting of low
dose, infrequent administration, self-administration and a
combination any of the foregoing.
[0175] In one embodiment, e.g., during an induction phase of
therapy, the dosing regimen provides an effective amount of an
anti-.alpha.4.beta.7 antibody or antigen-binding fragment in a
formulation described herein for inducing remission of an
inflammatory bowel disease in a human patient. In some embodiments,
the effective amount of the anti-.alpha.4.beta.7 antibody is
sufficient to achieve about 5 .mu.g/ml to about 60 .mu.g/ml, about
15 .mu.g/ml to about 45 .mu.g/ml, about 20 .mu.g/ml to about 30
.mu.g/ml, or about 25 .mu.g/ml to about 35 .mu.g/ml mean trough
serum concentration of the anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody or antigen-binding fragment thereof,
e.g., in a formulation described herein. Induction dosing can be
once, or a plurality of more than one dose, e.g., at least two
doses. During induction phase, a dose can be administered 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-.alpha.4.beta.7 antibody. In one
embodiment, induction dosing can be once at initiation of treatment
(day 0) and once at about two weeks after initiation of treatment.
In another embodiment, the induction phase duration is six weeks.
In another embodiment, the induction phase duration is six weeks
and a plurality of induction doses are administered during the
first two weeks.
[0176] In some embodiments, e.g., when initiating treatment of a
patient with severe inflammatory bowel disease (e.g., in patients
who have failed anti-TNF.alpha. 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 induction 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.
[0177] 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 .mu.g/mL, about 7 to about 20
.mu.g/mL, about 5 to about 10 .mu.g/mL, about 10 to about 20
.mu.g/mL, about 15 to about 25 .mu.g/mL or about 9 to about 13
.mu.g/mL of anti-.alpha.4.beta.7 antibody. In another embodiment,
the dosing regimen e.g., during a maintenance phase of therapy,
maintains a mean steady state trough serum concentration of about
20 to about 30 .mu.g/mL, about 20 to about 55 .mu.g/mL, about 30 to
about 45 .mu.g/mL, about 45 to about 55 .mu.g/mL or about 35 to
about 40 .mu.g/mL of anti-.alpha.4.beta.7 antibody.
[0178] 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
tolerance to the anti- .alpha.4.beta.7 antibody. A less frequent
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
thereafter. In one embodiment, the maintenance regimen includes a
dose every 8 weeks. In an embodiment, wherein a patient on a one
dose every eight weeks maintenance regimen experiences a return of
one or more disease symptoms, e.g., has a relapse, the dosing
frequency can be increased, e.g., to once every 4 weeks.
[0179] 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.
[0180] The dosing regimen can be optimized to induce a clinical
response and clinical remission in the inflammatory bowel disease
of the patient. In some embodiments, the dosing regimen does not
alter the ratio of CD4 to CD8 in cerebrospinal fluid of patients
receiving treatment.
[0181] 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.
[0182] In some aspects, a durable clinical response, for example, a
clinical response 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.
[0183] In one embodiment, the dosing regimen comprises an initial
dose of 300 mg, a second subsequent dose of 300 mg about two weeks
after the initial dose, a third subsequent dose of 300 mg at about
six weeks after the initial dose, followed by a fourth and
subsequent doses of 300 mg every four weeks or every eight weeks
after the third subsequent dose.
[0184] In some embodiments, the method of treatment, dose or dosing
regimen reduces the likelihood that a patient will develop a HAHA
response to the anti-.alpha.4.beta.7 antibody. The development of
HAHA, e.g., as measured by antibodies reactive to the
anti-.alpha.4.beta.7 antibody, can increase the clearance of the
anti-.alpha.4.beta.7 antibody, e.g., reduce the serum concentration
of the anti-.alpha.4.beta.7 antibody, e.g., lowering the number of
anti-.alpha.4.beta.7 antibody bound to .alpha.4.beta.7 integrin,
thus making the treatment less effective. In some embodiments, to
prevent HAHA, the patient can be treated with an induction regimen
followed by a maintenance regimen. In some embodiments, there is no
break between the induction regimen and the maintenance regimen. In
some embodiments, the induction regimen comprises administering a
plurality of doses of anti-.alpha.4.beta.7 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
mg/kg, at least 3 mg/kg, at least 5 mg/kg, at least 8 mg/kg, at
least 10 mg/kg or about 2 to about 6 mg/kg, or frequent initial
administrations, e.g., about once per week, about once every two
weeks or about once every three weeks, of the standard dose when
beginning therapy with an anti-.alpha.4.beta.7 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., .ltoreq.125,
of anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody.
[0185] The formulation may be administered to an individual (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
.alpha.4.beta.7 integrin to its ligands is administered. In such an
embodiment, an agent, e.g., a monoclonal antibody, such as an
anti-MAdCAM (e.g., anti-MAdCAM-1) or an anti-VCAM-1 monoclonal
antibody can be administered. In another embodiment, the additional
agent inhibits the binding of leukocytes to an endothelial ligand
in a pathway different from the .alpha.4.beta.7 pathway. Such an
agent can inhibit the binding, e.g. of chemokine (C--C motif)
receptor 9 (CCR9)-expressing lymphocytes to thymus expressed
chemokine (TECK or CCL25) or an agent which prevents the binding of
LFA-1 to intercellular adhesion molecule (ICAM). For example, an
anti-TECK or anti-CCR9 antibody or a small molecule CCR9 inhibitor,
such as inhibitors disclosed in PCT publication WO03/099773 or
WO04/046092, or anti-ICAM-1 antibody or an oligonucleotide which
prevents expression of ICAM, is administered in addition to a
formulation of the present invention. In yet another embodiment, an
additional active ingredient (e.g., an anti-inflammatory compound,
such as sulfasalazine, azathioprine, 6-mercaptopurine,
5-aminosalicylic acid containing anti-inflammatories, another
non-steroidal anti-inflammatory compound, a steroidal
anti-inflammatory compound, or antibiotics commonly administered
for control of IBD (e.g. ciprofloxacin, metronidazole), or another
biologic agent (e.g. TNF alpha antagonists) can be administered in
conjunction with a formulation of the present invention.
[0186] In an embodiment, the dose of the co-administered medication
can be decreased over time during the period of treatment by the
formulation comprising the anti-.alpha.4.beta.7 antibody. For
example, a patient being treated with a steroid (e.g. prednisone,
prednisolone) at the beginning, or prior to, treating with the
anti-.alpha.4.beta.7 antibody formulation would undergo a regimen
of decreasing doses of steroid beginning as early as 6 weeks of
treatment with the anti-.alpha.4.beta.7 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-.alpha.4.beta.7 antibody formulation. In one aspect, by about
16-24 weeks of treatment with the anti-.alpha.4.beta.7 antibody
formulation, the steroid dose can be eliminated. In another
example, a patient being treated with an anti-inflammatory
compound, such as 6-mercaptopurine at the beginning, or prior to,
treating with the anti-.alpha.4.beta.7 antibody formulation would
undergo a regimen of decreasing doses of anti-inflammatory compound
similar to the tapering regimen for steroid dosing as noted
above.
[0187] 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
administration can comprise a step of converting the formulation to
a liquid state. In one aspect, a dry formulation can be
reconstituted, e.g., by a liquid as described above, for use in
injection, e.g. intravenous, intramuscular or subcutaneous
injection. In another aspect, a solid or dry formulation can be
administered topically, e.g., in a patch, cream, aerosol or
suppository.
[0188] The invention also relates to a method for treating a
disease associated with leukocyte infiltration of tissues
expressing the molecule MAdCAM (e.g., MAdCAM-1). The method
comprises administering to a patient in need thereof an effective
amount of an anti-.alpha.4.beta.7 antibody formulation of the
invention. In an embodiment, the disease is graft versus host
disease. In some embodiments, the disease is a disease associated
with leukocyte infiltration of tissues as a result of binding of
leukocytes expressing .alpha.4.beta.7 integrin to gut-associated
endothelium expressing the molecule MAdCAM (e.g., MAdCAM-1). In
other embodiments, the disease is gastritis (e.g., eosinophilic
gastritis or autoimmune gastritis), pancreatitis, or
insulin-dependent diabetes mellitus. In yet other embodiments, the
disease is cholecystitis, cholangitis, or pericholangitis.
[0189] The invention also relates to a method for treating
inflammatory bowel disease in a patient. In one embodiment, the
method comprises administering to the patient an effective amount
of an anti-.alpha.4.beta.7 antibody formulation of the invention.
In some embodiments, the inflammatory bowel disease is ulcerative
colitis or Crohn's disease. In other embodiments, the inflammatory
bowel disease is Celiac disease, enteropathy associated with
seronegative arthropathies, microscopic or collagenous colitis,
gastroenteritis (e.g., eosinophilic gastroenteritis), or
pouchitis.
[0190] In some embodiments, treatment with an anti-.alpha.4.beta.7
antibody does not alter the ratio of CD4:CD8 lymphocytes. CD4:CD8
ratios can be measured in blood, lymph node aspirate, and
cerebro-spinal fluid (CSF). The CSF CD4+:CD8+ lymphocyte ratios in
healthy individuals are typically greater than or equal to about 1.
(Svennings son 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.
[0191] Articles of Manufacture
[0192] In another aspect, the invention is an article of
manufacture which contains the pharmaceutical formulation of the
present invention and provides instructions for its use. The
article of manufacture comprises a container. Suitable containers
include, for example, bottles, 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 directions for use. In another
embodiment, the formulation can be prepared for self-administration
and/or contain instructions for self-administration. In one aspect,
the container holding the formulation may be a single-use vial. In
another aspect, the container holding the formulation may be a
multi-use vial, which allows for 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 commercial and
user standpoint, including other buffers, diluents, filters,
needles, syringes and package inserts with instructions for use as
noted in the previous section.
[0193] Clinical and Quality Analysis
[0194] In another aspect, the invention is a method for determining
that a pharmaceutical formulation meets product quality standards.
The method may comprise evaluation of a lyophilized pharmaceutical
formulation (e.g., humanized anti-.alpha.4.beta.7 antibody)
comprising inspecting the formulation to assess appearance,
determining reconstitution time, determining moisture content of
lyophilized formulation, measuring aggregates in lyophilized
formulation, measuring fragmentation, measuring
oxidation/deamidation, and optionally assessing biological activity
and potency, wherein achievement of pre-determined standards
demonstrates product is indicated for clinical use.
[0195] Acceptable quality levels include .ltoreq.5.0% moisture,
.ltoreq.40 minutes reconstitution time, pH 6.3.+-.0.3 of
reconstituted liquid, 54.0 to 66.0 mg/ml antibody concentration,
.gtoreq.55.0% major isoform by CEX, .gtoreq.96.0% monomer by SEC,
.ltoreq.2.5% high molecular weight (aggregates), .gtoreq.90% H+L
chains by SDS-PAGE, 60-140% of the reference standard adhesion.
[0196] The invention will be more fully understood 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.
[0197] Development Protocol for Making Formulation
[0198] A. Anti-.alpha.4.beta.7 Antibody Solution
[0199] Bottles of frozen, high concentration anti-.alpha.4.beta.7
antibody preparation (vedolizumab, 50 mM histidine, 125 mM
arginine, 0.06% polysorbate 80, pH 6.3) are thawed at room
temperature for 16-24 hours. Thawed bottles are pooled into a
stainless steel compounding vessel and mixed. The preparation 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-.alpha.4.beta.7 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.
[0200] B. Lyophilization
[0201] Anti-.alpha.4.beta.7 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.degree. C. in a
lyophilizer. After loading all vials and closing the door, the
shelf temperature is lowered to freeze the solution, about
-45.degree. C. After 3 hours at this temperature, the temperature
of the shelves is raised to -20.degree. C. for annealing. After
annealing for four hours, the temperature of the shelves is lowered
to re-freeze the solution, about -45.degree. C. After equilibration
of the vials to this temperature, 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.degree. C.
Primary drying proceeds until the all of the crystalline ice has
sublimed from the vials. Then the shelf temperature is raised to
27.degree. C. for secondary drying for 16 hours, until the moisture
is approximately less than 2.5% of the lyophilized formulation.
When secondary drying is complete, nitrogen gas is backfilled into
the chamber until ambient pressure is reached. The vials are
stoppered and removed from the lyophilizer.
[0202] C. Storage and Use of Lyophilized Anti-.alpha.4.beta.7
Antibody
[0203] Lyophilized vials of anti-.alpha.4.beta.7 antibody are
stored at -70.degree. C., -20.degree. C., 2-8.degree. C. or
25.degree. C. for desired periods of time. When ready for use, a
vial is equilibrated to room temperature. Then the contents of the
vial are reconstituted 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 removed
using a syringe and is added and added to an IV bag for IV infusion
to a patient.
EXEMPLIFICATION
Example 1
Comparative Data for Varying % Sugar and Amino Acids in Lyophilized
Formulations
[0204] 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 arginine to protein, and
the molar amount of histidine buffer. Histidine and arginine are
known not to crystallize 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.degree. C., 150 mT and Secondary Drying at 20.degree. 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). FIG. 6A shows
the predictive models for changes in Percent Monomer, Percent
Aggregates, and Percent Major Isoform when stored at 40.degree. 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 examined, histidine did not affect the stability of the
formulation. All formulations had 1-2% moisture during storage.
TABLE-US-00001 TABLE 1 Change in Percent Monomer when stored at
5.degree. C., 25.degree. C./60% RH, and 40.degree. C./75% RH for 3
months. Percent Monomer was measured using Size Exclusion
Chromatography (SEC). % Monomer by SEC 25.degree. C. 40.degree. C.
Formulation 5.degree. C. 60% RH 75% RH 60 mg/mL vedolizumab + t = 0
3 mo 3 mo 3 mo 25 mM histidine, 75 mM 98.1 98.1 97.8 96.5 arginine,
2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM
98.0 98.2 98.0 97.5 arginine, 4% sucrose, 0.05% polysorbate 80, pH
6.9 50 mM histidine, 125 mM 98.0 98.3 98.1 97.4 arginine, 2%
sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM 98.0
98.3 98.1 97.4 arginine, 4% sucrose, 0.05% polysorbate 80, pH 6.9
50 mM histidine, 125 mM 98.7 98.4 98.4 98.1 arginine, 6% sucrose,
1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM
98.7 98.3 98.1 98.3 arginine, 9% sucrose, 0.06% polysorbate 80, pH
6.3
TABLE-US-00002 TABLE 2 Change in Percent Aggregates when stored
5.degree. C., 25.degree. C./60% RH, and 40.degree. C./75% RH for 3
months. Percent Monomer was measured using Size Exclusion
Chromatography (SEC). % Aggregates by SEC 25.degree. C. 40.degree.
C. Formulation 5.degree. C. 60% RH 75% RH 60 mg/mL vedolizumab + t
= 0 3 mo 3 mo 3 mo 25 mM histidine, 75 mM 0.42 0.53 0.89 1.99
arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine,
75 mM 0.41 0.51 0.62 1.15 arginine, 4% sucrose, 0.05% polysorbate
80, pH 6.9 50 mM histidine, 125 mM 0.42 0.47 0.60 1.23 arginine, 2%
sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM 0.36
0.44 0.52 0.82 arginine, 4% sucrose, 0.05% polysorbate 80, pH 6.9
50 mM histidine, 125 mM 0.53 0.49 0.51 0.56 arginine, 6% sucrose,
1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM
0.51 0.51 0.59 0.56 arginine, 9% sucrose, 0.06% polysorbate 80, pH
6.3
TABLE-US-00003 TABLE 3 Change in Percent Major Isoform when stored
at 5.degree. C., 25.degree. C./60% RH, and 40.degree. C./75% RH for
3 months. Major Isoform was measured using Cation Exchange
Chromatography (CEX). % Major Isoform by CEX 25.degree. C.
40.degree. C. Formulation 5.degree. C. 60% RH 75% RH 60 mg/mL
vedolizumab + t = 0 3 mo 3 mo 3 mo 25 mM histidine, 75 mM 70.5 68.8
67.4 66.3 arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM
histidine, 75 mM 70.8 98.9 68.0 67.7 arginine, 4% sucrose, 0.05%
polysorbate 80, pH 6.9 50 mM histidine, 125 mM 70.5 68.9 67.8 66.5
arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine,
125 mM 70.6 68.9 68.0 67.4 arginine, 4% sucrose, 0.05% polysorbate
80, pH 6.9 50 mM histidine, 125 mM 69.6 69.5 69.3 67.4 arginine, 6%
sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM
histidine, 125 mM 69.5 69.3 69.2 68.1 arginine, 9% sucrose, 0.06%
polysorbate 80, pH 6.3
[0205] FIG. 6A shows the predicted models based on the statistical
analysis of 40.degree. C. data from Tables 1-3. The model for
change in percent monomer per month at 40.degree. C. by SEC
analysis is -3.10+(0.386)*pH+0.000516*((moles of sugar+moles
arginine)/moles of protein)). The model for change in percent
aggregate per month at 40.degree. 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.degree. 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 predictive
models and the outer lines show the 95% confidence limit for the
predictive models.
[0206] FIG. 6B shows alternative models based on the statistical
analysis of 40.degree. C. data from Tables 1-3 when the input
factors are pH, sugar:protein molar ratio, and arginine:protein
molar ratio. The model for change in percent monomer per month at
40.degree. 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.degree. C. by SEC analysis is 2.35-(0.244)*pH-0.000727*((moles
of sugar)/(moles of protein)-0.00102*((moles of arginine)/(moles of
protein)). The model for change in percent major isoform per month
at 40.degree. C. by CEX analysis is
-2.92+(0.210)*pH+0.00164*((moles of sugar)/)/(moles of
protein)-0.000220*((moles of arginine)/(moles of protein)). 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
[0207] Stability Data
[0208] Three primary stability batches of the formulation (Batch A,
B, and C) were tested for stability after storage at the prescribed
storage condition (5 and 25.degree. C./60% RH for up to 24 months).
All three batches contain the same liquid formulation that was
lyophilized: 60 mg/mL anti-.alpha.4.beta.7 antibody, 50 mM
histidine, 125 mM arginine, 10% sucrose, 0.06% polysorbate 80, pH
6.3. For Batch A, 3.5 mL of solution was filled into 20 mL vials
and lyophilized, for Batches B and C, 5.52 mL of solution was
filled into 20 mL vials and lyophilized.
[0209] In a separate study, a single drug formulation of 60 mg/ml
anti-.alpha.4.beta.7 antibody, 50 mM histidine, 125 mM arginine,
10% sucrose, 0.06% polysorbate 80, pH 6.3 was lyophilized in two
volumes, 3.5 ml and 9.5 ml, respectively, to yield Batches R and S
for stability samples, which were analyzed over 38 months. Blanks
are NT (not tested).
[0210] The data (Tables 4-19) showed that the antibody formulations
remained stable when stored for up to 38 months at 5.degree. C. and
up to 30 months at 25.degree. C./60% RH. All product attributes
remained within the specifications through the 38 month time
point.
TABLE-US-00004 TABLE 4 Change in Percent Monomer by SEC when stored
at 5.degree. C. 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 98.8 99.2 3 99.8 99.1
99.1 98.8 98.8 6 99.8 99.8 99.8 98.9 99.0 9 99.1 99.2 99.2 99.2
99.1 12 99.4 99.0 99.0 98.8 98.9 15 99.4 99.1 99.1 18 99.5 99.4
99.4 98.9 98.9 24 99.4 99.2 99.2 99.0 99.0 30 99.2 99.2 38 99.3
99.3
TABLE-US-00005 TABLE 5 Change in Percent Aggregates by SEC when
stored at 5.degree. C. Time (months) Batch A Batch B Batch C Batch
R Batch S 0 0.1 0.1 0.1 0.2 0.2 1 0.1 0.2 0.2 0.2 0.1 3 0.1 0.2 0.2
0.2 0.2 6 0.2 0.2 0.2 0.2 0.2 9 0.1 0.2 0.2 0.2 0.2 12 0.2 0.2 0.2
0.2 0.2 15 0.2 0.2 0.2 18 0.2 0.2 0.2 0.2 0.2 24 0.2 0.2 0.2 0.2
0.2 30 0.2 0.2 38 0.2 0.2
TABLE-US-00006 TABLE 6 Change in Percent Major Isoform by CEX when
stored at 5.degree. C. Time (months) Batch A Batch B Batch C Batch
R Batch S 0 68.6 69.9 69.5 71.7 71.6 1 67.5 68.9 68.8 71.2 72.0 3
68.7 68.8 68.7 70.4 70.3 6 67.7 68.2 68.2 71.9 71.9 9 70.0 68.3
67.8 69.2 69.7 12 67.8 68.3 68.1 70.8 70.9 15 66.9 67.5 67.5 18
67.4 67.0 66.7 71.0 70.8 24 68.1 69.6 69.1 71.3 70.9 30 68.5 68.6
38 73.6 73.1
TABLE-US-00007 TABLE 7 Change in Percent Acidic Isoforms by CEX
when stored at 5.degree. 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 15 22.7 22.3 22.8
18 22.8 22.3 22.6 21.1 21.5 24 21.7 22.1 22.9 20.6 20.7 30 22.8
23.2 38 18.9 19.1
TABLE-US-00008 TABLE 8 Change in Percent Basic Isoforms by CEX when
stored at 5.degree. 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 6 9.5 8.7 8.2 7.0 6.7 9 10.2 9.6 9.3 9.0 8.4 12 9.3
10.3 9.9 8.0 7.9 15 10.4 10.1 9.7 18 9.8 10.7 10.7 7.9 7.7 24 10.2
8.3 8.1 8.1 8.3 30 8.7 8.2 38 7.5 7.7
TABLE-US-00009 TABLE 9 Change in % (H + L) by Reduced-SDS Page when
stored at 5.degree. C. Time (months) Batch A Batch B Batch C Batch
R Batch S 0 98 98 98 96 96 1 98 94 98 98 98 3 98 98 98 98 98 6 98
97 97 97 97 9 97 97 97 98 98 12 98 96 97 98 98 15 97 98 97 18 98 97
97 99 99 24 98 98 98 99 99 30 97 97 38 99 99
TABLE-US-00010 TABLE 10 Change in Binding Efficacy when stored at
5.degree. C. Time (months) Batch A Batch B Batch C Batch R Batch S
0 107 106 105 93 102 1 106 106 103 103 111 3 101 109 108 91 98 6 97
106 105 114 121 9 100 93 88 102 102 12 103 101 87 119 116 15 105 90
94 18 86 101 96 95 104 24 92 82 95 81 101 30 87 94 38 89 91
TABLE-US-00011 TABLE 11 Change in % Moisture by KF when stored at
5.degree. C. Time (months) Batch A Batch B Batch C Batch R Batch S
0 0.5 0.6 0.6 0.8 1.0 1 0.5 0.4 0.6 3 0.5 0.6 0.6 6 0.6 0.7 0.5 0.8
1.3 12 0.6 0.6 0.7 0.9 0.9 24 0.5 0.7 0.7 0.9 0.9 30 0.7 0.7
TABLE-US-00012 TABLE 12 Change in Percent Monomer by SEC when
stored at 25.degree. 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 99.0 98.6 98.5 6 99.8 99.7 99.7 98.9 98.9 9 99.0
99.1 99.1 99.1 99.1 12 99.3 98.9 98.9 98.8 98.9 15 99.3 99.0 99.0
18 99.4 99.3 99.3 98.7 98.9 24 99.2 99.1 99.1 98.9 98.9 30 99.0
99.0
TABLE-US-00013 TABLE 13 Change in Percent Aggregates by SEC when
stored at 25.degree. C./60% RH Time (months) Batch A Batch B Batch
C Batch R Batch S 0 0.1 0.1 0.1 0.2 0.2 1 0.2 0.2 0.2 0.2 0.2 3 0.2
0.3 0.2 0.3 0.3 6 0.2 0.3 0.3 0.2 0.2 9 0.2 0.3 0.3 0.2 0.2 12 0.2
0.2 0.2 0.3 0.3 15 0.3 0.3 0.3 18 0.3 0.3 0.3 0.3 0.2 24 0.3 0.3
0.3 0.3 0.2 30 0.4 0.3
TABLE-US-00014 TABLE 14 Change in Percent Major Isoform by CEX when
stored at 25.degree. C./60% RH Time (months) Batch A Batch B Batch
C Batch R Batch S 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 67.8 71.5 71.1 9 69.3
67.5 66.3 68.6 69.0 12 66.7 67.5 67.4 70.1 70.2 15 66.2 66.6 66.8
18 66.1 65.8 64.9 70.0 70.3 24 66.7 68.4 68.2 70.6 70.1 30 67.2
67.2
TABLE-US-00015 TABLE 15 Change in Percent Acidic Isoforms by CEX
when stored at 25.degree. C./60% RH 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 21.8 22.2
21.4 21.6 3 21.7 22.2 22.8 21.8 22.0 6 22.6 22.9 23.5 21.1 21.4 9
19.9 22.1 23.1 21.8 21.8 12 23.0 21.4 22.0 21.3 21.3 15 22.5 22.1
22.7 18 22.6 22.1 22.6 21.3 21.5 24 21.7 21.9 22.6 20.7 20.7 30
22.7 23.2
TABLE-US-00016 TABLE 16 Change in Percent Basic Isoforms by CEX
when stored at 25.degree. C./60% RH Time (months) Batch A Batch B
Batch C Batch R Batch S 0 8.5 9.3 9.1 8.1 7.8 1 10.8 9.8 9.2 7.4
7.3 3 10.3 9.3 9.0 7.8 7.7 6 11.5 9.3 8.7 7.4 7.5 9 10.8 10.4 10.6
9.7 9.3 12 10.3 11.1 10.7 8.7 8.5 15 11.3 11.2 10.6 18 11.2 12.1
12.5 8.7 8.2 24 11.6 9.7 9.1 8.7 9.2 30 10.2 9.6
TABLE-US-00017 TABLE 17 Change in % (H + L) by Reduced-SDS Page
when stored at 25.degree. C./60% RH Time (months) Batch A Batch B
Batch C Batch R Batch S 0 98 98 98 96 96 1 98 98 98 98 98 3 97 98
98 98 98 6 97 97 97 97 97 9 97 97 97 98 98 12 98 96 96 98 98 15 97
97 97 18 98 97 97 99 99 24 98 97 98 99 99 30 97 98
TABLE-US-00018 TABLE 18 Change in Binding Efficacy when stored at
25.degree. C./60% RH Time (months) Batch A Batch B Batch C Batch R
Batch S 0 107 106 105 93 102 1 115 103 109 3 92 113 100 96 94 6 109
89 97 101 114 9 97 89 85 97 102 12 83 91 123 15 96 91 96 18 106 123
87 92 102 24 103 82 90 98 94 30 84 114
TABLE-US-00019 TABLE 19 Change in % Moisture by KF when stored at
25.degree. C./60% RH Time (months) Batch A Batch B Batch C Batch R
Batch S 0 0.5 0.6 0.6 0.8 1.0 1 0.5 0.6 0.5 3 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 0.6 1.1 1.0 30 0.8
0.7
[0211] Cation Exchange Chromatography (CEX)
[0212] 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-.alpha.4.beta.7 antibody
formulations and determine the charge composition of the antibody
species. Acidic Isoforms elute before the Major Isoform and Basic
Isoforms elute after the Major Isoform.
[0213] 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 conditions, there was no trend of reducing
the % Major Isoform below 55.0%.
[0214] Size Exclusion Chromatography (SEC)
[0215] SEC is performed using an analytical SEC column (Tosoh
Bioscience, LLC, King of Prussia, Pa.). The mobile phase is a
phosphate-buffered saline solution and the absorbance is monitored
at 280 nm.
[0216] 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 lowering the % Monomer below
96.0%. Similarly, the % Aggregates remained .ltoreq.2.5% for all
batches at all listed storage conditions.
[0217] SDS-PAGE Assay
[0218] SDS-PAGE is performed using an Invitrogen (Carlsbad, Calif.)
Tris-Glycine gel, 4-20% for reducing condition and 4-12% for
non-reducing condition. The reconstituted antibody formulation
sample is diluted in liquid formulation buffer then diluted one to
two with Tris-Glycine SDS Sample Buffer (2.times., Invitrogen)
either with 10% 2-mercaptoethanol (reducing sample buffer) or
without 2-mercaptoethanol (non-reducing sample buffer). Samples are
briefly heated and loaded in comparison with a molecular 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 % IgG for non-reduced
gels.
[0219] Stability data generated using a Reduced SDS-PAGE assay are
presented in
[0220] Tables 9 and 17. No noticeable changes were observed for the
% Heavy+Light (H+L) chains at all storage conditions listed for all
stability lots. The banding pattern was similar to that of the
reference standard and % (H+L) remained at a level .gtoreq.90%.
[0221] Binding Efficacy
[0222] 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 antibody. After a
further wash, the cells are fixed and suspended in FACS reagent for
analysis by flow cytometry (Becton Dickinson Franklin Lakes, N.J.);
also see U.S. Pat. No. 7,147,851.
[0223] Binding efficacy of vedolizumab was measured relative to the
Reference Standard and reported as % Reference Standard and EC50.
Stability data is presented 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 storage conditions listed.
[0224] Moisture by Karl Fischer
[0225] The formulation is titrated 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 storage conditions.
[0226] Capillary Isoelectric Focusing (cIEF)
[0227] 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 available ampholytes. A useful
combination is a mixture of 3-10 and 5-8 PHARMALYTE.TM. (GE
Healthcare, Piscataway, N.J.).
Example 3
Modeling the Scale-Up of the Lyophilization Process
[0228] 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 formulation solids content was
varied from 9 to 27% by including in the loads a formulation which
was 0.5.times., 1.0.times. and 1.5.times. of the target
formulation. These formulations had similar T.sub.g'. With higher %
solids, the primary drying time increased. In addition, at higher
solids content, the product temperature increased due to larger
R.sub.p. The load also has an effect on both stages of drying (FIG.
8).
Example 4
Non-Clinical Safety Study
[0229] A study was designed to compare the effect of natalizumab
and vedolizumab on immune surveillance of the CNS in Rhesus EAE.
Eight animals were dosed with a placebo control, once weekly. Seven
animals were dosed at 30 mg/kg, once weekly, with natalizumab.
Seven animals 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.
[0230] 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 (p<0.05) delayed the onset of clinical symptoms
of
[0231] EAE as compared to placebo control. It inhibited the
incidence of EAE and the magnitude of clinical scores. (FIG. 9)
[0232] Vedolizumab did not prevent infiltration of the CSF by
leukocytes, T lymphocytes (helper T lymphocytes, cytotoxic T
lymphocytes), B lymphocytes, natural killer cells, or monocytes. In
contrast, natalizumab inhibited infiltration of the CSF
[0233] 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) inhibition in brain infiltrates and
demyelination was measured by histology.
[0234] The .alpha.4.beta.7 integrin was saturated by vedolizumab
during the investigation, as shown by a competitive binding assay
between vedolizumab dosed in vivo and an analytical
anti-.alpha.4.beta.7 monoclonal antibody added ex vivo. The
analytical anti-.alpha.4.beta.7 mAb does not bind to memory helper
T lymphocytes in animals dosed with vedolizumab. The lack of effect
of vedolizumab in the CNS is therefore due to the
gastrointestinal-tropic biology of the .alpha.4.beta.7
integrin.
[0235] In summary, vedolizumab (an .alpha.4.beta.7 antagonist) does
not inhibit EAE. In contrast, natalizumab (.alpha.4.beta.1 and
.alpha.4.beta.7 antagonist) does inhibit EAE. The .alpha.4.beta.1
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 .alpha.4.beta.1
integrin and impair immune surveillance of the CNS in Rhesus
EAE.
Example 5
Phase I Clinical Study with Vedolizumab
[0236] 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.degree. C. and up to 3 months at -20.degree. C.)) and 10
subjects received placebo. Of the 39 subjects who received
vedolizumab, 8 subjects each received a dose at 0.2, 2.0, 6.0, and
10.0 mg/kg and 7 subjects received vedolizumab at 0.5 mg/kg. All 49
subjects completed the study.
[0237] There were no notable differences across vedolizumab cohorts
for any demographic or baseline characteristic. Mean age ranged
from 35.4 to 51.0 years; individual subject ages ranged from 21 to
63 years.
[0238] PK Results
[0239] Vedolizumab was administered as a 30-minute intravenous
infusion of 0.2 to 10.0 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 cohorts, indicating dose proportionality for this parameter.
The dose-normalized area under the serum drug concentration value
from time zero to infinity (AUC.sub.0-inf) increased with
increasing dose up to 2.0 mg/kg, indicating there was a nonlinear
increase in AUC.sub.0-inf with increasing dose over the lower range
of doses administered in this study. Thereafter, AUC.sub.0-inf
increased proportionally with dose, indicating linearity of
AUC.sub.0-inf over the dose range 2.0 to 10.0 mg/kg. The increase
in AUC.sub.0-inf was approximately 2.4-fold higher than expected at
the 10.0 mg/kg dose compared with the 0.2 mg/kg dose.
[0240] 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,
distribution volume increased, and, consequently, the terminal
elimination half-life 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 fraction of clearance of vedolizumab at higher
doses.
[0241] 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-US-00020 TABLE 20 Overview of Vedolizumab PK by Dose Cohort
Following IV Administration of 0.2-10.0 mg/kg Vedolizumab in
Healthy Subjects (PK Analysis Set) VDZ Geometric Parameter dose N
Mean SD Mean % CV Median Min Max C.sub.max 0.2 4 5.65 0.629 5.62
11.1 5.45 5.13 6.56 (.mu.g/mL) mg/kg 0.5 4 10.6 2.09 10.4 19.7 10.6
8.07 13.1 mg/kg 2.0 7 59.3 11.6 58.4 19.6 58.4 47.6 78.4 mg/kg 6.0
6 151 19.1 150 12.6 157 120 168 mg/kg 10.0 7 243 22.1 243 9.07 242
213 281 mg/kg AUC.sub.0-tlast 0.2 4 31.6 4.98 31.3 15.8 31.6 25.7
37.5 (day*.mu.g/mL) mg/kg 0.5 4 127 48.0 119 37.9 129 70.9 178
mg/kg 2.0 7 964 147 955 15.2 972 772 1170 mg/kg 6.0 6 3090 749 3020
24.2 2830 2360 4100 mg/kg 10.0 7 4870 624 4840 12.8 4750 4120 5870
mg/kg AUC.sub.0-inf 0.2 4 39.5 5.79 39.1 14.7 40.2 31.7 45.7
(day*.mu.g/mL) mg/kg 0.5 4 134 48.9 127 36.5 134 79.2 188 mg/kg 2.0
7 979 146 969 14.9 993 784 1180 mg/kg 6.0 6 3100 750 3030 24.2 2840
2390 4110 mg/kg 10.0 7 4880 637 4850 13.0 4750 4130 5920 mg/kg
V.sub.z(L) 0.2 4 4.02 0.151 4.02 3.76 4.03 3.83 4.18 mg/kg 0.5 4
4.92 0.620 4.89 12.6 4.66 4.52 5.84 mg/kg 2.0 7 3.34 0.665 3.28
19.9 3.23 2.29 4.27 mg/kg 6.0 6 2.98 0.644 2.92 21.6 2.98 2.06 3.98
mg/kg 10.0 7 2.89 1.02 2.73 35.2 2.98 1.49 4.58 mg/kg CL (L/day)
0.2 4 0.413 0.042 0.412 10.1 0.395 0.388 0.476 mg/kg 0.5 4 0.310
0.106 0.297 34.3 0.291 0.212 0.446 mg/kg 2.0 7 0.165 0.018 0.164
10.7 0.162 0.145 0.194 mg/kg 6.0 6 0.140 0.031 0.136 22.0 0.145
0.083 0.166 mg/kg 10.0 7 0.140 0.024 0.139 16.9 0.135 0.103 0.171
mg/kg t.sub.1/2 (day 0.2 4 6.79 0.736 6.76 10.8 6.95 5.79 7.47
mg/kg 0.5 4 11.7 2.83 11.4 24.2 11.4 9.09 14.8 mg/kg 2.0 7 14.1
2.67 13.9 18.9 14.3 10.6 17.5 mg/kg 6.0 6 15.1 3.15 14.8 20.9 14.0
11.9 20.3 mg/kg 10.0 7 14.8 7.38 13.7 49.8 12.5 8.26 30.7 mg/kg
Abbreviations: AUC.sub.0-inf = area under the drug
concentration-time curve, extrapolated to infinity; AUC.sub.0-tlast
= 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;
C.sub.max = maximum drug concentration; t.sub.1/2 = terminal
half-life; V.sub.z = volume of distribution based on the terminal
phase.
[0242] After reaching C.sub.max, 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 nonlinear fashion.
[0243] The C.sub.max 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
parameter. The dose-normalized AUC.sub.0-inf increased with
increasing dose up to 2.0 mg/kg, indicating there was a nonlinear
increase in AUC.sub.0-inf with increasing dose over the lower range
of doses administered in this study. Thereafter, AUC.sub.0-inf
increased proportionally with dose, indicating linearity of
AUC.sub.0-inf over the dose range 2.0 to 10.0 mg/kg. The increase
in AUC.sub.0-inf was approximately 2.4-fold higher than expected at
the 10.0 mg/kg dose compared with the 0.2 mg/kg dose.
[0244] PD Results
[0245] 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-US-00021 TABLE 21 Overview of Vedolizumab Pharmacodynamics,
Percent Inhibition of % Act- 1.sup.+ [CD4.sup.+ CD45RO.sup.high],
by Dose Cohort Following IV Administration of 0.2-10.0 mg/kg
Vedolizumab in Healthy Subjects (PD Analysis Set) VDZ Geometric
Parameter dose N Mean SD Mean % CV Median Min Max E.sub.max 0.2 4
99.6 0.387 99.6 0.388 99.6 99.1 100 (% Inhibition) mg/kg 0.5 4 99.5
0.599 99.5 0.602 99.5 98.9 100 mg/kg 2.0 6 99.9 0.172 99.9 0.172
100 99.6 100 mg/kg 6.0 6 100 0.000 100 0.000 100 100 100 mg/kg 10.0
6 99.7 0.326 99.7 0.327 99.8 99.3 100 mg/kg AUEC.sub.0-inf 0.2 4
4030 1010 3920 25.2 4090 2760 5160 (% Inhibition*d) mg/kg 0.5 4
6430 1450 6300 22.6 6530 4860 7810 mg/kg 2.0 6 13200 623 13200 4.72
12900 12800 14500 mg/kg 6.0 6 16700 3030 16500 18.1 16300 13300
20100 mg/kg 10.0 6 19300 644 19300 3.33 19600 18200 19900 mg/kg
AUEC.sub.0-inf = area under the drug effect versus time curve from
time 0 to the time of the last non-zero concentration; E.sub.max =
maximum drug effect
TABLE-US-00022 TABLE 22 Overview of Vedolizumab Pharmacodynamics,
Percent Inhibition of % MADCAM.sup.+ [CD4.sup.+ CD45RO.sup.high],
by Dose Cohort Following IV Administration of 0.2-10.0 mg/kg
Vedolizumab in Healthy Subjects (PD Analysis Set) VDZ Geometric
Parameter dose N Mean SD Mean % CV Median Min Max E.sub.max 0.2 4
99.2 0.537 99.2 0.542 99.4 98.4 99.6 (% Inhibition) mg/kg 0.5 4
99.6 0.323 99.6 0.324 99.5 99.3 100 mg/kg 2.0 6 99.7 0.365 99.7
0.366 99.7 99.2 100 mg/kg 6.0 6 99.8 0.279 99.8 0.280 100 99.4 100
mg/kg 10.0 6 100 0.000 100 0.000 100 100 100 mg/kg AUEC.sub.0-inf
0.2 4 4000 576 3970 14.4 4210 3160 4440 (% Inhibition*d) mg/kg 0.5
4 6770 1400 6660 20.6 6840 5170 8230 mg/kg 2.0 6 13000 796 13000
6.12 13000 11700 13900 mg/kg 6.0 6 16200 3320 15900 20.5 15800
11800 20000 mg/kg 10.0 6 17700 1330 17700 7.5 17700 16500 19000
mg/kg AUEC.sub.0-inf = area under the drug effect versus time curve
from time 0 to the time of the last non-zero concentration;
E.sub.max = maximum drug effect
[0246] Vedolizumab inhibited the PD parameters, Act-1 and
MAdCAM-1-Fc, nearly maximally at all time points where vedolizumab
was measurable in serum. Once vedolizumab concentrations decreased
below the limit of detection of the assay, the inhibition of Act-1
and MAdCAM-1-Fc returned to approximately the baseline level.
[0247] In some subjects who developed HAHA to vedolizumab, a faster
loss of .alpha.4.beta.7 receptor saturation was observed as
compared to the HAHA-negative subjects in the respective dose
level.
[0248] Safety Results
[0249] 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.
[0250] Immunogenicity/Human Antihuman Antibody (HAHA) Formation
[0251] One (10%) subject in the placebo group and 21 (54%) subjects
in the combined vedolizumab 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 observed previously with vedolizumab.
Nineteen of the 22 vedolizumab-treated subjects who were
HAHA-positive had neutralizing HAHA present.
TABLE-US-00023 TABLE 23 Overview of Human Antihuman Antibodies
Findings: Safety Population 0.2 0.5 2.0 6.0 10.0 mg/kg mg/kg mg/kg
mg/kg mg/kg Combined Placebo VDZ VDZ VDZ VDZ VDZ VDZ N = 10 N = 8 N
= 7 N = 8 N = 8 N = 8 N = 39 Subjects Tested 10 8 7 8 8 8 39 Any
HAHA Positive, n (%) 1 (10) 6 (75) 4 (57) 2 (25) 3 (38) 6 (75) 21
(54) Highest HAHA 1 (10) 4 (50) 2 (29) 2 (25) 3 (38) 6 (75) 17 (44)
Titer < 125, n(%) Highest HAHA 0 2 (25) 2 (29) 0 0 0 4 (10)
Titer .gtoreq. 125, n(%) Any Neutralizing HAHA 0 5 (63) 4 (57) 2
(25) 3 (38) 5 (63) 19 (49) Positive, n(%) Highest Neutralizing 0 3
(38) 2 (29) 2 (25) 3 (38) 5 (63) 15 (38) HAHA Titer < 125, n(%)
Highest Neutralizing 0 2 (25) 2 (29) 0 0 0 4 (10) HAHA Titer
.gtoreq. 125, n(%)
[0252] One subject in the placebo group and 11 subjects in the
vedolizumab group were persistently HAHA-positive.
TABLE-US-00024 TABLE 24 Overall Human Antihuman Antibody Status
(Safety Population) 0.2 0.5 2.0 6.0 10.0 mg/kg mg/kg mg/kg mg/kg
mg/kg Combined Placebo VDZ VDZ VDZ VDZ VDZ VDZ N = 10 N = 8 N = 7 N
= 8 N = 8 N = 8 N = 39 HAHA negative.sup.a 9 (90).sup. 2 (25) 3
(43) 6 (75) 5 (63) 2 (25) 18 (46) n(%) Isolated HAHA.sup.b 0 2 (25)
1 (14) 1 (13) 1 (13) 5 (63) 10 (26) n(%) Persistent HAHA.sup.c 1
(10).sub.-- 4 (50) 3 (43) 1 (13) 2 (25) 1 (13) 11 (28) n(%)
.sup.aHAHA Negative: Subjects with no positive HAHA results
.sup.bIsolated HAHA: Subjects with only 1 positive HAHA sample with
titer < 25 .sup.cPersistent HAHA: Subjects with 2 or more
positive HAHA samples, or 1 positive sample with titer .gtoreq.
25
[0253] Conclusions
[0254] 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 selection for phase 3
pivotal trials in inflammatory bowel disease.
[0255] Vedolizumab demonstrated dose proportionality over the
tested dose range for the Cmax parameter; however, dose-dependent
changes in AUC0-inf, CL, Vz, and t1/2 were observed from 0.2 to 2.0
mg/kg, suggesting nonlinear PK behavior of vedolizumab. 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.
[0256] Vedolizumab inhibited the PD parameters, Act-1 and
MAdCAM-1-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-1-Fc returned to approximately
the baseline level.
[0257] In some subjects who developed HAHA to vedolizumab, a faster
clearance of vedolizumab and loss of .alpha.4.beta.7 receptor
saturation was observed as compared to the HAHA-negative subjects
within the respective dose level.
[0258] Vedolizumab was well-tolerated. No deaths, SAEs, or AEs
leading to discontinuation of study drug administration occurred
during the study, nor were any dose-toxicity relationships
observed. No systemic opportunistic infections (including PML) or
neoplasms were reported.
[0259] Unlike nonspecific .alpha.4 antagonists, vedolizumab was not
associated with lymphocytosis or mean increases in circulating
eosinophils, basophils, or monocytes, nor was there any evidence of
depletion of lymphocytes.
[0260] 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 vedolizumab may minimize
clinically significant HAHA formation.
[0261] 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 CD4:CD8 Ratio
[0262] Healthy subjects ages 18-45 were treated with a single 450
mg dose of vedolizumab reconstituted from a lyophilized formulation
of 10% sucrose and diluted into an infusion 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.
[0263] 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. N Engl J Med. 2003;348(1):15-23); and also because at 5
weeks, a 450-mg dose of vedolizumab 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.
[0264] Approximately 15 mL CSF was obtained from each subject for
immunophenotyping. CSF samples were included for analyses if they
met the following criteria: .ltoreq.10 RBCs/.mu.L per sample (to
minimize peripheral blood contamination); negative CSF culture
result; adequate T-lymphocyte numbers in each flow cytometry
sample; and no detection of serum antibodies to vedolizumab.
[0265] Week 5 median (34.80 .mu.g/mL) and individual subject serum
vedolizumab concentrations (range 24.9-47.9 .mu.g/mL) were higher
than projected steady-state trough concentration (.about.24
.mu.g/mL) for the phase 3 dose regimen. A high degree (>90%) of
.alpha.4.beta.7 receptor saturation was observed at week 5 as
measured by MAdCAM-1-Fc, indicating vedolizumab's saturation of its
target at the time of endpoint assessment.
[0266] Vedolizumab was not detected in any CSF sample (detection
limit=0.125 .mu.g/mL).
[0267] Effect on CD4+ and CD8+ T Lymphocyte Numbers and Ratio
[0268] Vedolizumab did not significantly reduce CD4+:CD8+ ratio
(Table 25). None of the subjects had a postdose CD4+:CD8+ ratio
<1 (p <0.0001 (1-sided t-test)). Vedolizumab did not
significantly reduce the number of CD4+ or CD8+ T lymphocytes in
CSF. In addition, there were no significant changes in CSF % CD4+
and % CD8+ T lymphocytes (Table 26). Also, no significant changes
in peripheral blood WBC, CD4+ and CD8+ memory T lymphocytes (Table
27) were observed.
TABLE-US-00025 TABLE 25 Effect of Treatment on CSF CD4+:CD8+ Ratio
(Evaluable Population, n = 13) CD4+:CD8+ Ratio Baseline Week 5
Difference.dagger. CD4+:CD8+ ratio 3.59 (0.273) 3.60 (0.265)* 0.01
(0.197) Mean (SE) Range 1.53-5.67 1.42-5.15 90% 2-sided CI for
3.00-4.19 3.132, 4.077 ratio 90% 2-sided CI for -0.337, 0.363
difference CI = confidence interval *p < 0.0001 (one sided one
sample t-test for H0: .mu. < 1 vs H1: .mu. >= 1).
.dagger.Difference is defined as week 5 ratio minus baseline
ratio
TABLE-US-00026 TABLE 26 Treatment Effect on CSF CD4+ and CD8+
Lymphocyte Count (Evaluable Population, n = 13) Baseline Week 5
CD4+ as % 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-US-00027 TABLE 27 Peripheral Blood Memory T Lymphocytes (RO+)
Counts (Evaluable Population, n = 13) Baseline Week 5 Mean (SD)
Mean (SD) CD4+CD45RO+ 27.85 (4.98) 27.06 (5.02) CD8+CD45RO+(%)
11.24 (3.40) 10.78 (2.98)
[0269] Summary
[0270] 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+ subsets in peripheral blood. Saturation
of the target (.alpha.4.beta.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.
[0271] These results are consistent with vedolizumab's lack of
effect on both physiologic CNS immune surveillance and pathologic
CNS inflammation of monkeys (See Example 4).
Example 7
Long-Term Clinical Experience with Vedolizumab for the Treatment of
IBD
[0272] A phase 2 open-label safety extension study was completed to
assess the long-term pharmacokinetics (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 confirmed endoscopically and/or
histopathologically and/or radiologically within 36 months of
screening.
[0273] All patients received an intravenous dosing regimen of
either 2 mg/kg or 6 mg/kg of 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.degree. C. and up to 3 mo -20.degree.
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
colitis patients that had participated in an earlier clinical
trial.
[0274] Efficacy/quality of life (QoL); partial Mayo score (PMS),
Crohn's disease activity index (CDAI), and Inflammatory Bowel
Disease Questionnaire (IBDQ) were used to assess the results of the
study.
[0275] PK Results
[0276] Mean pre-infusion vedolizumab concentrations were dose
proportional, and remained steady and detectable throughout the
study.
[0277] PD Results
[0278] Receptors (% ACT-1
[0279] +[CD4+CD45RO HIGH] and % MADCAM+[CD4+CD45RO HIGH] were
almost fully inhibited throughout the study period at all dose
levels.
[0280] Partial Mayo Score
[0281] 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 pronounced 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.
[0282] Crohn's Disease Activity Index
[0283] CD patients' mean CDAI decreased from 294.6 at baseline to
237.7 at Day 43, and continued to decrease through day 155
(156.1).
[0284] IBDQ
[0285] 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 treatment-naive
ulcerative colitis patients and ulcerative colitis rollover
patients.
[0286] C-Reactive Protein
[0287] Both ulcerative colitis rollover and Crohn's disease
patients showed decreased mean CRP levels through day 155 and then
leveled off. Treatment-naive ulcerative colitis patients had a
lower mean CRP level at baseline than ulcerative colitis rollover
patients (2.28 v. 7.09). Mean CRP levels of the treatment-naive
ulcerative colitis patients remained relatively constant at all
time points assessed.
[0288] Other Safety Results
[0289] No systematic opportunistic infections (including 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.
[0290] Conclusions
[0291] 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 IBDQ scores, was generally safe and well tolerated, and
demonstrated acceptable immunogenicity.
Example 8
Induction of Response and Remission in Patients with Moderate to
Severely Active Crohn's Disease
[0292] A randomized, double blind, placebo controlled multi-center
study was completed to evaluate the induction 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 TNF.alpha.
antagonist failure patients at week 6 (after 2 doses--0 and 2
weeks) and at week 10 (after 3 doses). The study consisted of 416
patients, 75% of whom were TNF.alpha. antagonist failures, and 25%
of whom were TNF.alpha. naive. Demographics and concomitant IBD
medication were balanced across treatment groups. Baseline disease
characteristics were also balanced across treatment groups, except
for baseline disease activity.
[0293] The primary endpoint designated for the study was week 6
remission (%) in anti-TNF-.alpha. antagonist failure population.
The key secondary endpoints that were evaluated (sequential testing
procedure) were: week 6 remission (%) in overall population, week
10 remission (%) in anti-TNF-.alpha. antagonist failure and overall
population (using Hochberg procedure), week 6 and 10 sustained
remission (%) in anti-TNF-.alpha. antagonist failure and overall
population (using Hochberg procedure), and week 6 enhanced response
(%) in anti-TNF-.alpha. antagonist failure population.
TABLE-US-00028 TABLE 28 Baseline CDAI: Placebo Vedolizumab p-value
TNF ITT: Mean 306.1 (55.43) 316.1 (52.63) 0.0945 (Std Dev) Overall
ITT: Mean 301.3 (54.97) 313.9 (53.17) 0.0153 (Std Dev)
TABLE-US-00029 TABLE 29 Induction Study Results: Primary and Key
Secondary Endpoints TNF ITT (N = 315) Overall ITT (N = 416) PLA VDZ
Diff P- PLA VDZ Diff P- Endpoints N = 157 V = 158 (RR) value N =
207 N = 209 (RR) value Primary Wk 6 Remission 12.1% 15.2% 3.0%
(1.2) 0.4332 1st Secondary Wk 6 12.1% 19.1% 6.9% (1.6) 0.0478
Remission 2nd Secondary 12.1% 26.6% 14.4% (2.2) 0.0012 .sup. 13%
28.7% 15.5% (2.2) <0.0001 Wk 10 Remission Sustained Remission
8.3% 12.0% 3.7% (1.4) 0.2755 8.2% 15.3% 7% (1.9) 0.0249 (both Wk
6&10) Enhanced Response 22.3% 39.2% 16.9% (1.8) 0.0011
(CDAI100)
TABLE-US-00030 TABLE 30 Results in Anti-TNF-.alpha. Antagonist
Naive Patients (n = 101, 24% of overall) Placebo Vedolizumab
Difference % % % 95% Cl Remission Week 6 12 31.4 19.1 (3.3, 35.0)
Remission Week 10 16 35.3 19.2 (2.4, 35.8)
TABLE-US-00031 TABLE 31 Study Results: Clinical Remission at Weeks
6 and 10, Key Subgroup-Previous Tx Failures, ITT Overall Subgroup
Variable Placebo VDZ Diff 95% Cl Any prior anti- N 156 155 TNF
failure (75% Wk 6 Rem 12.8 14.8 2 (-5.7, 9.7) of ITT) (%) Wk 10 Rem
12.8 26.5 13.6 (4.9, 22.3) (%) Prior N 45 44 immunomodulator Wk 6
Rem 11.1 31.8 20.7 (-0.5, 39.7) failure but not (%) anti-TNF
failure Wk 10 Rem 15.6 31.8 16.3 (-1.1, 33.6) (21% ITT) (%) Prior N
5 9 corticosteroid Wk 6 Rem 0 33.3 33.3 (-23.9, 75.7) failure only
(3% (%) ITT) Wk 10 Rem 0 44.4 44.4 (-13.4, 85.3) (%)
[0294] The study showed that TNF-.alpha. antagonist failure
patients required 3 doses for induction of remission. Remission
rates in TNF-.alpha. antagonist failure patients increased between
week 6 and week 10, but only for the vedolizumab group (not
placebo). Remission rates for TNF-.alpha. antagonist naive patients
did not increase substantially between week 6 and 10. Of the
TNF-.alpha. antagonist failure population with a high degree of
disease severity, 43% never responded to a TNF-.alpha. antagonist,
and 45% lost response.
Example 9
Induction and Maintenance of Response and Remission in Patients
with Moderately to Severely Active Ulcerative Colitis
[0295] 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. Demographic and baseline disease
characteristics were comparable across all treatment groups.
[0296] 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, 10%
sucrose, at pH 6.3, with an endpoint at 6 weeks after 2 doses of
vedolizumab.
[0297] 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, diagnosed with
moderately to severely active ulcerative colitis; demonstrated,
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
conventional therapies for IBD. The endpoint of this study was at
52 weeks, analyzing the induction responder population. Both phases
of the trial met their primary endpoints, namely, clinical response
in induction and clinical remission in maintenance.
[0298] Blood samples were collected to measure concentrations of
vedolizumab during the study. The mean serum concentration of
vedolizumab at the end of the induction phase was 20 to 30
.mu.g/mL. The mean vedolizimab trough serum concentrations at
steady state after 30 min IV infusion of 300 mg dose administration
were between 9 to 13 .mu.g/mL for the q8wks (8 weeks) regimen and
between 35 to 40 .mu.g/mL for the q4wks (4 weeks) regimen. At the
end of infusion, the vedolizimab median plasma concentrations were
between 98 and 101 .mu.g/mL for the q8ks regimen and around 129 and
137 .mu.g/mL for the q4 wks.
[0299] Summaries of the responses of the induction and maintenance
studies are provided in Tables 32-35. A significantly greater
proportion of vedolizumab-treated patients achieved 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-TNF.alpha. 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 analyses through week 6,
rates of adverse events (AEs), serious AEs, and adverse events
leading to study discontinuation were higher in the placebo group
than vedolizumab group. A significantly greater proportion of
vedolizumab patients than placebo 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 population had prior anti-TNF.alpha. failure.
Clinical remission and durable clinical response rates were greater
with vedolizumab 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 AEs, and serious infections were
similar between vedolizumab and placebo groups. No increase in
rates of opportunistic or enteric infections was observed in the
vedolizumab group.
TABLE-US-00032 TABLE 32 Induction Study Results-Primary and Key
Secondary Endpoints Efficacy Endpoints Placebo Vedolizumab
Difference/RR P value Clinical 25.5% 47.1% 21.7%/1.8 <0.0001
Response (%) Clinical 5.4% 16.9% 11.5%/3.1 0.0010 Remission (%)
Mucosal 24.8% 40.9 16.1%/1.6 0.0013 Healing (%)
TABLE-US-00033 TABLE 33 Maintenance Study Results-Primary and Key
Secondary Endpoints Differ- ence/RR Efficacy Placebo VDZ Q8 VDZ Q4
Q8 vs. Pb Endpoint N = 126 N = 122 N = 125 Q4 vs. Pb P value
Clinical 15.9 41.8 44.8 26.1/2.7 <0.0001 Remission (%) 29.1/2.8
<0.0001 Durable 23.8 56.6 52.0 32.8/2.4 <0.0001 Response (%)
28.5/2.2 <0.0001 Mucosal 19.8 51.6 56.0 32.0/2.6 <0.0001
Healing (%) 36.3/2.8 <0.0001 Durable 8.7 20.5 24.0 11.8/2.4
0.0090 Remission (%) 15.3/2.8 0.0011 Corticosteroid- 13.9 31.4 45.2
17.6/2.3 0.0133 free Remission n = 72 n = 70 N = 73 31.4/3.3
<0.0001 (%)
TABLE-US-00034 TABLE 34 Induction Study: Clinical Response and
Remission at 6 Weeks in Patients with Prior Anti-TNF-.alpha.
Antagonist Failure and Without Anti-TNF Exposure, ITT Population
Patients with Prior Anti-TNF-.alpha. Antagonist Failure (39%)
Placebo Vedolizumab Endpoint N = 63 N = 82 Difference 95% Cl
Clinical 20.6 39.0 18.4 3.9, 32.9 Response (%) Clinical 3.2 9.8 6.6
-9.8, 22.8 Remission (%) Patients Without Anti-TNF-.alpha.
Antagonist Exposure (55%) Placebo Vedolizumab N = 76 N = 130
Difference 95% Cl Clinical 26.3 53.1 26.8 13.7, 39.9 Response (%)
Clinical 6.6 23.1 16.5 2.4, 30.2 Remission (%)
TABLE-US-00035 TABLE 35 Clinical Remission and Durable Clinical
Response at 52 Weeks: Patients with Prior Anti-TNF-.alpha.
Antagonist Failure or Without Anti-TNF-.alpha. Antagonist Exposure
ITT Population Patients with Prior Anti-TNF-.alpha. Antagonist
Failure (32%) Difference Q8 wks vs VDZ VDZ Placebo Placebo Q8 Wks
Q4 Wks Q4 wks vs. Endpoint N = 38 N = 43 N = 40 Placebo 95% Cl
Clinical 5.3 37.2 35.0 31.9 10.3, 51.4 remission (%) 29.7 7.4, 49.4
Durable 15.8 46.5 42.5 30.7 11.8, 49.6 Clinical 26.7 7.5, 45.9
Response (%) Patients without Anti-TNF-.alpha. Antagonist Exposure
(60%) Difference Q8 wks vs. VDZ VDZ Placebo Placebo Q8 wks Q4 wks
Q4 wks vs. N = 79 N = 72 N = 73 Placebo 95% Cl Clinical 19.0 45.8
47.9 26.8 12.4, 41.2 Remission (%) 29.0 14.6, 43.3 Durable 26.6
65.3 56.2 38.7 24.0, 53.4 Clinical 29.6 14.6, 44.6 Response (%)
Example 10
Induction and Maintenance of Response and Remission in Patients
with Moderately to Severely Active Crohn's Disease
[0300] 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 Crohn's Disease. Demographic and baseline disease
characteristics were comparable across all treatment groups.
[0301] 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, 10%
sucrose, at pH 6.3, with an endpoint at 6 weeks after 2 doses of
vedolizumab.
[0302] The maintenance study, using the same formulation and route
of administration as the induction study, compared placebo against
vedolizumab dosed every four weeks, and placebo against vedolizumab
dosed every eight weeks. The endpoint of this study was at 52
weeks, analyzing the induction responder population.
[0303] Surprisingly, 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
significantly 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 (AEs), serious AEs, and serious
infections were similar between vedolizumab and placebo groups. No
increase in rates of opportunistic or enteric infections was
observed in the vedolizumab group.
TABLE-US-00036 TABLE 36 Induction Study Results-Primary and
Secondary Endpoints Placebo Vedolizumab Adjusted Endpoints N = 148
N = 220 Difference/RR P value Clinical 6.8% 14.5% 7.8%/2.1 0.0206
Remission (%) Enhanced 25.7% 31.4% 5.7%/1.2 0.2322 Response (%)
Mean CRP -3.6 -2.9 0.9288 Change N = 147 N = 220 (.mu.g/mL)
TABLE-US-00037 TABLE 37 Maintenance Study Results-Primary and Key
Secondary Endpoints Adj. Differ- ence/RR Efficacy Placebo VDZ Q8
VDZ Q4 Q8 vs. Pb P Endpoint N = 153 N = 154 N = 154 Q4 vs. Pb value
Clinical 21.6 39.0 36.4 17.4/1.8 0.0007 Remission (%) 14.7/1.7
0.0042 Enhanced 30.1 43.5 45.5 13.4/1.4 0.0132 Response (%)
15.3/1.5 0.0053 Corticosteroid- 15.9 31.7 28.8 15.9/2.0 0.0154 free
Remission N = 82 N = 82 N = 80 12.9/1.8 0.0450 (%) Durable 14.4
21.4 16.2 7.2/1.5 0.1036 Remission (%) 2.0/1.1 0.6413
TABLE-US-00038 TABLE 38 Clinical Remission and Enhanced Response at
6 Weeks in Patients with Prior Anti-TNF-.alpha. Antagonist Failure
and Without Anti-TNF Exposure, ITT Population Patients with Prior
Anti-TNF-.alpha. Antagonist Failure (48%) Placebo Vedolizumab
Endpoint N = 70 N = 105 Difference 95% Cl Clinical 4.3 10.5 6.2
(-9.1, 21.3) Remission (%) Enhanced 22.9 23.8 1.0 (-11.8, 13.7)
Response (%) Patients Without Anti-TNF-.alpha. Antagonist Exposure
(50%) Placebo Vedolizumab N = 76 N = 130109 Difference 95% Cl
Clinical 9.2 17.4 8.2 (-1.4, 17.9) Remission (%) Enhanced 30.3 42.2
11.9 (-1.9, 25.8) Response (%)
TABLE-US-00039 TABLE 39 Clinical Remission and Enhanced Response at
52 Weeks: Patients with Prior Anti-TNF-.alpha. Antagonist Failure
or Without Anti-TNF-.alpha. Antagonist Exposure ITT Population
Patients with Prior Anti-TNF-.alpha. Antagonist Failure (51%)
Difference Q8 wks vs VDZ VDZ Placebo Placebo Q8 Wks Q4 Wks Q4 wks
vs. Endpoint N = 78 N = 82 N = 77 Placebo 95% Cl Clinical 12.8 28.0
27.3 15.2 (3.0, 27.5) remission (%) 14.5 (2.0, 26.9) Enhanced 20.5
29.3 37.7 8.8 (-4.6, 22.1) Response (%) 17.1 (3.1, 31.2) Patients
without Anti-TNF-.alpha. Antagonist Exposure (45%) Difference Q8
wks vs. VDZ VDZ Placebo Placebo Q8 wks Q4 wks Q4 wks vs. N = 71 N =
66 N = 71 Placebo 95% Cl Clinical 26.8 51.1 46.5 24.8 (8.9, 40.6)
Remission (%) 19.7 (4.2, 35.2) Enhanced 38.0 60.6 53.5 22.6 (6.3,
38.9) Response (%) 15.5 (-0.7, 31.7)
TABLE-US-00040 TABLE 40 Summary of Sequences SEQ ID NO: Sequence
Shown Description 1 FIG. 1 DNA encoding heavy chain of humanized
anti-.alpha.4.beta.7 immunoglobulin 2 FIG. 1 Amino acid sequence of
heavy chain of humanized anti-.alpha.4.beta.7 immunoglobulin 3 FIG.
2 DNA encoding the light chain of humanized anti- .alpha.4.beta.7
immunoglobulin 4 FIG. 2 Amino acid sequence of light chain of
humanized anti-.alpha.4.beta.7 immunoglobulin 5 FIG. 3 Mature
humanized light chain of LDP-02 6 FIG. 4 Generic human kappa light
chain constant region 7 FIG. 4 Generic murine kappa light chain
constant region 8 Referenced on page 30 CDR1 of heavy chain SYWMH
mouse ACT-1 antibody 9 Referenced on page 30 CDR2 of heavy chain
EIDPSESNTNYNQKFKG mouse ACT-1 antibody 10 Referenced on page 30
CDR3 of heavy chain GGYDGWDYAIDY mouse ACT-1 antibody 11 Referenced
on page 30 CDR1 of light chain RSSQSLAKSYGNTYLS mouse ACT-1
antibody 12 Referenced on page 30 CDR2 of light chain GISNRFS mouse
ACT-1 antibody 13 Referenced on page 30 CDR3 of light chain
LQGTHQPYT mouse ACT-1 antibody 14 FIG. 7 human GM607 CL antibody
kappa light chain variable region 15 FIG. 7 Human 21/28 CL antibody
heavy chain variable region
[0304] 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 departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
1511445DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 1gaattctcga gatcgatctc
accatgggat ggagctgtat catcctcttc ttggtagcaa 60cagctacagg tgtccactcc
caggtgcaat tggtgcagtc tggggctgag gttaagaagc 120ctggggcttc
agtgaaggtg tcctgcaagg gttctggcta caccttcacc agctactgga
180tgcattgggt gaggcaggcg cctggccaac gtctagagtg gatcggagag
attgatcctt 240ctgagagtaa tactaactac aatcaaaaat tcaagggacg
cgtcacattg actgtagaca 300tttccgctag cacagcctac atggagctct
ccagcctgag atctgaggac actgcggtct 360actattgtgc aagagggggt
tacgacggat gggactatgc tattgactac tggggtcaag 420gcaccctggt
caccgtcagc tcagcctcca ccaagggccc atcggtcttc cccctggcac
480cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc
aaggactact 540tccccgaacc ggtgacggtg tcgtggaact caggcgccct
gaccagcggc gtgcacacct 600tcccggctgt cctacagtcc tcaggactct
actccctcag cagcgtggtg accgtgccct 660ccagcagctt gggcacccag
acctacatct gcaacgtgaa tcacaagccc agcaacacca 720aggtggacaa
gaaagttgag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc
780cagcacctga actcgcgggg gcaccgtcag tcttcctctt ccccccaaaa
cccaaggaca 840ccctcatgat ctcccggacc cctgaggtca catgcgtggt
ggtggacgtg agccacgaag 900accctgaggt caagttcaac tggtacgtgg
acggcgtgga ggtgcataat gccaagacaa 960agccgcggga ggagcagtac
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc 1020accaggactg
gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag
1080cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca
caggtgtaca 1140ccctgccccc atcccgggat gagctgacca agaaccaggt
cagcctgacc tgcctggtca 1200aaggcttcta tcccagcgac atcgccgtgg
agtgggagag caatgggcag ccggagaaca 1260actacaagac cacgcctccc
gtgctggact ccgacggctc cttcttcctc tacagcaagc 1320tcaccgtgga
caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
1380aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt
aaataatcta 1440gagca 14452470PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 2Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr
Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp
Val Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60 Glu Trp Ile Gly Glu
Ile Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe
Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser 85 90 95 Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105
110 Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp
115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230
235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu 245 250 255 Leu Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355
360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu
Ser Pro Gly Lys 465 470 3751DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 3gaattctcga gatcgatctc accatgggat ggagctgtat
catcctcttc ttggtagcaa 60cagctacagg tgtccactcc gatgtagtga tgactcaaag
tccactctcc ctgcctgtca 120cccctggaga accagcttct atctcttgca
ggtctagtca gagtcttgca aagagttatg 180ggaacaccta tttgtcttgg
tacctgcaga agcctggcca gtctccacag ctcctcatct 240atgggatttc
caacagattt tctggggtgc cagacaggtt cagtggcagt ggttcaggga
300cagatttcac actcaagatc tcgcgagtag aggctgagga cgtgggagtg
tattactgct 360tacaaggtac acatcagccg tacacgttcg gacaggggac
caaggtggag atcaagcgta 420cggtggctgc accatctgtc ttcatcttcc
cgccatctga tgagcagttg aaatctggaa 480ctgcctctgt tgtgtgcctg
ctgaataact tctatcccag agaggccaaa gtacagtgga 540aggtggataa
cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca
600aggacagcac ctacagcctc agcagcaccc tgaccctgag caaagcagac
tacgagaaac 660acaaagtcta cgcctgcgaa gtcacccatc agggcctgag
ctcgcccgtc acaaagagct 720tcaacagggg agagtgttag tctagagcag c
7514238PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 4Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25 30 Thr Pro Gly Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Ala Lys
Ser Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro 50 55 60
Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser 65
70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys 100 105 110 Leu Gln Gly Thr His Gln Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235 5219PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 5Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Ala Lys Ser 20 25 30 Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gly
Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr
His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110 Arg Ala Asp Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 215 6107PRTHomo sapiens 6Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10
15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105 7107PRTMus sp. 7Arg Ala Asp Ala Ala Pro
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser
Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50
55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr
Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr
Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105 85PRTMus sp. 8Ser Tyr Trp Met His 1 5 917PRTMus sp. 9Glu
Ile Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10
15 Gly 1012PRTMus sp. 10Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp
Tyr 1 5 10 1116PRTMus sp. 11Arg Ser Ser Gln Ser Leu Ala Lys Ser Tyr
Gly Asn Thr Tyr Leu Ser 1 5 10 15 127PRTMus sp. 12Gly Ile Ser Asn
Arg Phe Ser 1 5 139PRTMus sp. 13Leu Gln Gly Thr His Gln Pro Tyr Thr
1 5 14111PRTHomo sapiens 14Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85
90 95 Leu Gln Thr Pro Gln Thr Phe Gly Gln Gly Lys Val Glu Ile Lys
100 105 110 15119PRTHomo sapiens 15Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp
Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Ser Gly Ser Asn Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
* * * * *