U.S. patent application number 11/817993 was filed with the patent office on 2009-04-30 for anti-m-csf antibody compositions.
This patent application is currently assigned to Pfizer, Inc.. Invention is credited to Corey M. Allan, James Carroll, Tapan K. Das, Sandeep Nema, David Zeng.
Application Number | 20090110681 11/817993 |
Document ID | / |
Family ID | 36646098 |
Filed Date | 2009-04-30 |
United States Patent
Application |
20090110681 |
Kind Code |
A1 |
Carroll; James ; et
al. |
April 30, 2009 |
Anti-M-CSF Antibody Compositions
Abstract
The present invention provides for compositions of anti-M-CSF
antibodies comprising a chelating agent and/or histidine. Also
provided are methods of treating M-CSF-mediated disorders with
pharmaceutical formulations of anti-M-CSF anti-bodies, including
inflammatory diseases and neoplasia disorders.
Inventors: |
Carroll; James;
(Chesterfield, MO) ; Das; Tapan K.; (Ballwin,
MO) ; Allan; Corey M.; (Manchester, MO) ;
Nema; Sandeep; (St. Louis, MO) ; Zeng; David;
(Chesterfield, MO) |
Correspondence
Address: |
PHARMACIA CORPORATION;GLOBAL PATENT DEPARTMENT
POST OFFICE BOX 1027
ST. LOUIS
MO
63006
US
|
Assignee: |
Pfizer, Inc.
New York
NY
|
Family ID: |
36646098 |
Appl. No.: |
11/817993 |
Filed: |
March 2, 2006 |
PCT Filed: |
March 2, 2006 |
PCT NO: |
PCT/US2006/007461 |
371 Date: |
December 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60659766 |
Mar 8, 2005 |
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60728165 |
Oct 19, 2005 |
|
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60752712 |
Dec 20, 2005 |
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60762456 |
Jan 26, 2006 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 11/00 20180101; A61P 21/00 20180101; A61P 31/04 20180101; A61P
35/04 20180101; A61P 5/50 20180101; A61P 9/14 20180101; A61P 11/06
20180101; A61P 17/06 20180101; A61P 35/00 20180101; A61P 19/02
20180101; C07K 2317/21 20130101; A61P 29/00 20180101; A61P 37/00
20180101; A61P 19/08 20180101; A61P 43/00 20180101; A61K 2300/00
20130101; A61P 21/04 20180101; A61K 9/19 20130101; A61P 27/02
20180101; A61P 13/10 20180101; A61K 47/183 20130101; A61P 37/06
20180101; A61P 1/18 20180101; A61K 39/3955 20130101; A61P 17/02
20180101; A61P 19/10 20180101; A61P 31/12 20180101; A61K 9/08
20130101; A61K 39/39591 20130101; A61K 47/26 20130101; A61P 3/10
20180101; A61P 1/04 20180101; A61P 31/18 20180101; A61P 9/10
20180101; A61K 2039/505 20130101; A61P 5/14 20180101; A61P 15/00
20180101; C07K 16/243 20130101; A61P 41/00 20180101; A61P 1/02
20180101; A61P 37/08 20180101; A61K 39/3955 20130101; A61P 1/14
20180101; A61P 3/02 20180101; A61P 1/16 20180101; A61P 11/04
20180101; A61P 25/00 20180101; A61P 7/06 20180101; A61P 31/00
20180101; A61P 37/02 20180101; A61P 7/02 20180101; A61P 35/02
20180101; A61P 21/02 20180101 |
Class at
Publication: |
424/139.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 19/02 20060101
A61P019/02 |
Claims
1. A composition comprising at least one chelating agent; and at
least one antibody comprising: an amino acid sequence that is at
least 90% identical to a heavy chain amino acid sequence shown in
SEQ ID NO: 2, and an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human M-CSF.
2. The composition according to claim 1, wherein the composition is
a liquid composition, the antibody is a human IgG2 antibody, and
the antibody does not comprise a signal sequence.
3. The composition according to claim 1, wherein the antibody
comprises a heavy chain amino acid sequence with at least 99%
sequence identity to SEQ ID NO: 2 and a light chain amino acid
sequence with at least 99% sequence identity to SEQ ID NO: 4.
4. The composition according to claim 1, wherein the antibody
comprises a heavy chain amino acid sequence comprising the variable
region of SEQ ID NO: 2 and a light chain amino acid sequence
comprising the variable region of SEQ ID NO: 4.
5. The composition according to claim 1, wherein the antibody
comprises an isolated human monoclonal IgG2 anti-M-CSF antibody
having the heavy and light chain amino acid sequences of antibody
8.10.3F.
6. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA.
7. The composition according to claim 1, wherein the composition
further comprises a buffer.
8. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises at least one buffer that is histidine.
9. The composition according to claim 1, wherein the composition
further comprises a buffer and a surfactant.
10. The composition according to claim 1, wherein the composition
further comprises a buffer, a surfactant, and a tonicity agent.
11. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises a buffer, a surfactant, and a tonicity agent.
12. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine, a surfactant, and a tonicity agent.
13. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine, polysorbate 80, and a tonicity agent.
14. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of a chelating agent;
and from about 1 mM to about 100 mM of histidine.
15. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of EDTA; and from
about 1 mM to about 100 mM of histidine.
16. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 1.0 millimolar of a chelating agent;
from about 1 mM to about 100 mM of a buffer; from about 0.01 mg/ml
to about 10 mg/ml of a surfactant; and from about 100 millimolar to
about 400 millimolar of a tonicity agent.
17. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 1.0 millimolar of EDTA; from about 1
mM to about 100 mM of a buffer; from about 0.01 mg/ml to about 10
mg/ml of a surfactant; and from about 100 millimolar to about 300
millimolar of a tonicity agent.
18. The composition according to claim 2, wherein the composition
comprises: from about 1 mg/ml to about 100 mg/ml of antibody; from
about 0.01 millimolar to about 1.0 millimolar of a chelating agent;
from about 1 mM to about 100 mM of histidine; from about 0.01 mg/ml
to about 2 mg/ml of a surfactant; and from about 1 mg/ml to about
to about 200 mg/ml of mannitol.
19. The composition according to claim 2, wherein the composition
comprises: about 10 mg/ml of antibody; about 0.02 mg/ml of a
chelating agent; about 10 mM histidine; about 0.2 mg/ml of
polysorbate 80; and about 45 mg/ml of mannitol.
20. The composition according to claim 1, wherein the composition
comprises: from about 50 mg/ml to about 100 mg/ml of antibody; from
about 0.01 mg/ml to about 1 mg/ml of EDTA; from about 5 mM to about
50 mM histidine; from about 0.1 mg/ml to about 2 mg/ml of
polysorbate 80; and from about 1 mg/ml to about 150 mg/ml of a
tonicity agent.
21. A composition comprising at least one monoclonal anti-M-CSF
antibody and a chelating agent, wherein the composition comprises
an amount of the chelating agent sufficient to stabilize the
composition when maintained at a temperature of about 40.degree. C.
for a period of at least about 26 weeks.
22. A composition comprising at least one anti-M-CSF antibody and a
chelating agent, wherein the molar concentration of the antibody
ranges from about 0.01 millimolar to about 2 millimolar and the
molar concentration of the chelating agent ranges from about 0.001
millimolar to about 5 millimolar, and wherein the molar ratio of
antibody to chelating agent ranges from about 0.002 to about
2000.
23. A process for preparing a composition comprising mixing at
least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and an amino acid sequence that is at least 90%
identical to a heavy chain amino acid sequence shown in SEQ ID NO:
2; with at least one chelating agent.
24. A method for the treatment of an M-CSF-mediated disorder in a
subject, comprising administering to the subject a therapeutically
effective amount of a liquid pharmaceutical composition comprising
at least one pharmaceutically acceptable chelating agent; and at
least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the antibody binds to human M-CSF.
25. The method according to claim 24, wherein the M-CSF-mediated
disorder is selected from the group consisting of neoplasia
disorders and inflammatory disorders.
Description
CROSS-REFERENCE TO RELATED PATENTS AND PATENT APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/659,766 filed Mar. 8, 2005; U.S.
Provisional Patent Application Ser. No. 60/728,165 filed Oct. 19,
2005; U.S. Provisional Patent Application Ser. No. 60/752,712 filed
Dec. 20, 2005; and U.S. Provisional Patent Application Ser. No.
60/762,456 filed Jan. 26, 2006, all of which are incorporated by
reference herein in their entireties.
BACKGROUND OF THE INVENTION
[0002] Macrophage colony stimulating factor (M-CSF) is a secreted
or a cell surface glycoprotein comprised of two subunits that are
joined by a disulfide bond with a total molecular mass varying from
40 to 90 kDa ((Stanley, et al., Mol. Reprod. Dev., 46:4-10 (1997)).
Similar to other colony stimulation factors, M-CSF is produced by
macrophages, monocytes, and human joint tissue cells, such as
chondrocytes and synovial fibroblasts, in response to proteins such
as interleukin-1 or tumor necrosis factor-alpha. M-CSF stimulates
the formation of macrophage colonies from pluripotent hematopoietic
progenitor stem cells (Stanley, et al., Mol. Reprod. Dev., 46:4-10
(1997)).
[0003] M-CSF is an important regulator of the function, activation,
and survival of monocytes/macrophages. A number of animal models
have confirmed the role of M-CSF in various diseases, including
rheumatoid arthritis and cancer. Macrophages comprise key effector
cells in rheumatoid arthritis. The degree of synovial macrophage
infiltration in rheumatoid arthritis has been shown to closely
correlate with the extent of underlying joint destruction. M-CSF,
endogenously produced in the rheumatoid joint by
monocytes/macrophages, fibroblasts, and endothelial cells, acts on
cells of the monocyte/macrophage lineage to promote their survival
and differentiation into bone destroying osteoclasts, and enhance
pro-inflammatory cellular functions such as cytotoxicity,
superoxide production, phagocytosis, chemotaxis and secondary
cytokine production. Antibodies to M-CSF have been used to treat
animal models of rheumatoid arthritis (Cambell, et al., J.
Leukocyte Biol. 68:144-150 (2000)). For example, U.S. Published
Application No. 20050059113 to Bedian, et al. describes anti-M-CSF
antibodies. Thus, there is a need in the art for formulations of
M-CSF antibodies that can be used to treat diseases, and in
particular such diseases as rheumatoid arthritis, cancer, and other
M-CSF-mediated diseases.
SUMMARY
[0004] In one aspect, the present invention provides a composition
comprising at least one antibody comprising an amino acid sequence
that is at least 90% identical to a light chain amino acid sequence
shown in SEQ ID NO: 4, and further comprising an amino acid
sequence that is at least 90% identical to a heavy chain amino acid
sequence shown in SEQ ID NO: 2, wherein the antibody binds to human
M-CSF; and further comprising a chelating agent.
[0005] The present invention also provides a liquid pharmaceutical
composition comprising at least one antibody comprising an amino
acid sequence that is at least 90% identical to a light chain amino
acid sequence shown in SEQ ID NO: 4, and further comprising an
amino acid sequence that is at least 90% identical to a heavy chain
amino acid sequence shown in SEQ ID NO: 2, wherein the antibody
binds to human M-CSF; and further comprising a chelating agent.
[0006] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent.
[0007] The present invention also provides a composition comprising
at least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the composition contains antibody in
amount of at least about 10 mg/ml and the antibody binds to human
M-CSF; and a chelating agent.
[0008] The present invention also provides a composition comprising
at least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the composition contains antibody in
amount of equal to or less than about 15 mg/ml and the antibody
binds to human M-CSF; and a chelating agent.
[0009] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF and the C-terminal lysine of the heavy chain of the
antibody is not present; and a chelating agent.
[0010] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent, wherein the composition
further comprises at least one or two excipients selected from the
group consisting of tonicity agents, surfactants and buffers.
[0011] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a pharmaceutically acceptable chelating agent,
wherein the composition further comprises a tonicity agent, a
surfactant and a buffer.
[0012] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent, wherein the composition
further comprises a tonicity agent selected from the group of
mannitol, trehalose and sucrose.
[0013] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and EDTA.
[0014] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF and the concentration of the antibody is from about
0.1 mg/ml to about 100 mg/ml; and EDTA.
[0015] The present invention also provides a composition comprising
at least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the antibody binds to human M-CSF; and
further comprising a chelating agent, wherein the composition is
administered to the subject parenterally.
[0016] The present invention also provides a composition comprising
at least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the antibody binds to human M-CSF; and
further comprising a chelating agent, wherein the composition is
administered to the subject intravenously.
[0017] The present invention also provides a composition comprising
at least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the antibody binds to human M-CSF; and
further comprising a chelating agent, wherein the composition
comprises from about 0.1 mg/ml to about 200 mg/ml of antibody; from
about 1 mM to about 100 mM of histidine; from about from about 0.01
mg/ml to about 10 mg/ml of polysorbate 80; from about 1 micromolar
to about 1.0 millimolar of EDTA; and from about 10 millimolar to
about 600 millimolar of a tonicity agent.
[0018] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent, and the antibody is stable
at a temperature of about 5.degree. C. for at least about 26
weeks.
[0019] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent, and the antibody is stable
at a temperature of about 25.degree. C. for at least about 26
weeks.
[0020] The present invention also provides a composition comprising
at least one antibody wherein the antibody comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 2 and a light chain amino
acid sequence comprising SEQ ID NO: 4, wherein the antibody binds
to human M-CSF; and a chelating agent, and the antibody is stable
at a temperature of about 40.degree. C. for at least about 26
weeks.
[0021] The present invention also provides a stable composition
comprising at least one monoclonal anti-M-CSF antibody and a
stabilizing amount of a chelating agent, wherein after the
composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between a fragment
chromatogram peak area for the stable composition comprising at
least one monoclonal anti-M-CSF antibody and the chelating agent,
and a fragment chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 1%; or the decrease
between a fragment chromatogram peak area for the stable
composition comprising at least one monoclonal anti-M-CSF antibody
and the chelating agent, and a fragment chromatogram peak area for
an isotonic composition lacking the chelating agent, is at least
about 0.5%.
[0022] The present invention also provides a method for stabilizing
at least one monoclonal anti-M-CSF antibody comprising the method
of forming a composition comprising the antibodies and a
stabilizing amount of a chelating agent, wherein after the
composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between an aggregate
chromatogram peak area for the stable composition comprising
monoclonal anti-M-CSF antibodies and the chelating agent, and an
aggregate chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 1%; or the decrease
between an aggregate chromatogram peak area for the stable
composition comprising monoclonal anti-M-CSF antibodies and the
chelating agent, and an aggregate chromatogram peak area for an
isotonic composition lacking the chelating agent, is at least about
0.5%.
[0023] The present invention also provides methods for stabilizing
at least one monoclonal anti-M-CSF antibody in a composition
comprising the method: forming a composition comprising the
antibody and at least one stabilizing chelating agent compound,
wherein the composition comprises a sufficient amount of the
stabilizing chelating agent to stabilize the composition during
storage for a period of about 26 weeks at a temperature of about
40.degree. C.; and wherein at the end of the storage period at
least one of the following conditions are satisfied: the amount of
aggregated antibodies is less than or equal to about 3.5% by peak
area of a chromatogram of the antibodies after chromatographic
separation; or the amount of antibody fragment formed by antibody
hydrolysis having a molecular weight ranging from about 10.5 kD to
about 11.5 kD is less than or equal to about 1.7% by peak area of a
chromatogram of the antibodies after chromatographic
separation.
[0024] The present invention also provides a method for analyzing
the stability of at least one anti-M-CSF antibody comprising
identifying an antibody fragment formed by antibody hydrolysis
having a molecular weight ranging from about 10.5 kD to about 11.5
kD polypeptide fragment in a composition comprising the anti-M-CSF
antibody by separating species by organic SE-HPLC, and identifying
the presence of the polypeptide fragment in the composition by
using ultraviolet detection at 214 nanometers.
[0025] The present invention also provides a method for detecting
the presence of a polypeptide fragment having a molecular weight of
between about 10 kD and about 12 kD in a composition comprising at
least one anti-M-CSF antibody antibody, the method comprising
separating species by organic SE-HPLC, and identifying the presence
of the polypeptide fragment in the composition by using ultraviolet
detection at 214 nanometers or at other suitable wavelengths, e.g.,
280 nm.
[0026] The present invention also provides a method for stabilizing
at least one anti-M-CSF antibody by combining the antibody in a
liquid composition with a chelating agent in an amount, which
reduces chemical or physical instability of the antibody.
[0027] The invention also provides a liquid pharmaceutical
composition comprising at least one anti-M-CSF antibody and a
chelating agent, wherein the molar concentration of the antibody
ranges from about 0.01 millimolar to about 2 millimolar and the
molar concentration of the chelating agent ranges from about 0.001
millimolar to about 5 millimolar, and wherein the molar ratio of
antibody to chelating agent ranges from about 0.002 to about 2000;
from about 0.01 to about 500; from about 0.05 to about 100; from
about 0.1 to about 50; from about 0.5 to about 10; from about 1 to
about 5; from about 1 to about 3; or about 1.6.
[0028] The present invention also provides a composition comprising
at least one anti-M-CSF antibody and a chelating agent, wherein the
molar concentration of the antibody ranges from about 0.01
millimolar to about 2 millimolar and the molar concentration of the
chelating agent ranges from about 0.001 millimolar to about 5
millimolar, and wherein the molar ratio of antibody to chelating
agent ranges from about 0.002 to about 2000; from about 0.01 to
about 500; from about 0.05 to about 100; from about 0.1 to about
50; from about 0.5 to about 10; from about 1 to about 5; or about
3.8.
[0029] The present invention also provides methods for the
treatment of an M-CSF-mediated disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
a liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a chelating
agent.
[0030] The present invention also provides a method for the
treatment of an M-CSF-mediated disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
a liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a chelating
agent, wherein the subject is in need of the treatment of a
M-CSF-mediated disorder.
[0031] The present invention also provides methods for the
treatment of an M-CSF-mediated disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
a liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a chelating
agent, wherein the M-CSF-mediated disorder is a neoplasia
disorder.
[0032] The present invention also provides methods for the
treatment of an M-CSF-mediated disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
a liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a chelating
agent, wherein the M-CSF-mediated disorder is osteoarthritis.
[0033] The present invention also provides methods for the
treatment of an M-CSF-mediated disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
a liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a chelating
agent, wherein the M-CSF-mediated disorder is rheumatoid
arthritis.
[0034] The present invention provides a liquid pharmaceutical
composition comprising at least one antibody comprising an amino
acid sequence that is at least 90% identical to a light chain amino
acid sequence shown in SEQ ID NO: 4, and further comprising an
amino acid sequence that is at least 90% identical to a heavy chain
amino acid sequence shown in SEQ ID NO: 2, wherein the antibody
binds to human M-CSF and the composition contains a concentration
of antibody that is at least about 10 mg/ml; and further comprising
a chelating agent.
[0035] The present invention provides compositions comprising at
least one antibody comprising an amino acid sequence that is at
least 90% identical to a light chain amino acid sequence shown in
SEQ ID NO: 4, and further comprising an amino acid sequence that is
at least 90% identical to a heavy chain amino acid sequence shown
in SEQ ID NO: 2, wherein the antibody binds to human M-CSF and the
composition contains a concentration of antibody that ranges from
about 50 mg/ml to about 100 mg/ml; and further comprising a
chelating agent.
[0036] The present invention also provides a process for preparing
a composition comprising mixing at least one anti-M-CSF antibody
having the heavy and light chain amino acid sequences of antibody
8.10.3F in solution, with a chelating agent.
[0037] The present invention also provides methods for preparing a
stable composition comprising mixing at least one monoclonal
anti-M-CSF antibody 8.10.3F with at least one chelating agents, in
an amount, which reduces chemical or physical instability of the
antibody.
[0038] The present invention also provides an article of
manufacture comprising a container which holds a mixture of at
least one anti-M-CSF antibody having the heavy and light chain
amino acid sequences of antibody 8.10.3F, and a chelating
agent.
[0039] The present invention also provides kits for preparing
compositions of at least one anti-M-CSF antibody comprising: a
first container comprising at least one anti-M-CSF antibody having
the heavy and light chain amino acid sequences of antibody 8.10.3F,
and a second container comprising a chelating agent.
[0040] The present invention also provides kits for preparing a
liquid pharmaceutical composition of at least one anti-M-CSF
antibody comprising: a first container comprising at least one
anti-M-CSF antibody having the heavy and light chain amino acid
sequences of antibody 8.10.3F in solution, and a second container
comprising a pharmaceutically acceptable chelating agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows a line graph that shows the percent
fragmentation in SDS-PAGE reduced gels with anti-M-CSF antibody
8.10.3F compositions stored for six weeks at 40.degree. C.;
[0042] FIG. 2 shows a line graph that shows the percent major IEF
band in IEF gels with anti-M-CSF antibody 8.10.3F compositions
stored for six weeks at 40.degree. C.;
[0043] FIG. 3 shows a line graph that shows the percent aggregation
determined from SE-HPLC data with anti-M-CSF antibody 8.10.3F
compositions stored for six weeks at 40.degree. C.;
[0044] FIG. 4 shows a line graph that shows the percent aggregation
determined from SE-HPLC data with anti-M-CSF antibody 8.10.3F
compositions stored for 26 weeks at 40.degree. C.;
[0045] FIG. 5 shows a line graph that shows the percent
fragmentation (approximately 11 kD) determined from organic SE-HPLC
data with anti-M-CSF antibody 8.10.3F compositions stored for up to
26 weeks at 40.degree. C.;
[0046] FIG. 6 shows a line graph that shows the percent
fragmentation from SDS-PAGE reduced gels with anti-M-CSF antibody
8.10.3F compositions stored for 26 weeks at 40.degree. C.;
[0047] FIG. 7 shows a line graph that shows the percentage of
remaining antibody monomer from SDS-PAGE non-reduced gels with
anti-M-CSF antibody 8.10.3F compositions stored for 26 weeks at
40.degree. C.;
[0048] FIG. 8 shows an organic SE-HPLC chromatogram (shown expanded
in y-axis) of anti-M-CSF antibody 8.10.3F compositions 11 and 9
stored for 26 weeks at 40.degree. C.;
[0049] FIG. 9 shows a photograph of a reduced SDS-PAGE gel of
anti-M-CSF antibody 8.10.3F compositions stored for six weeks at
40.degree. C.;
[0050] FIG. 10 shows an organic SE-HPLC chromatogram of anti-M-CSF
antibody 8.10.3F in formulation 1 (top) stored at 40.degree. C. for
6 weeks compared to a control sample (bottom).
[0051] FIG. 11 shows an SE-HPLC chromatogram for anti-M-CSF
antibody 8.10.3F stored in formulation 11 for 26 weeks at
40.degree. C.
[0052] FIG. 12, comprising FIGS. 12A-12D, shows the nucleic acid
and amino acid sequence for anti-M-CSF antibody 8.11.3F. FIG. 12A
shows the full-length nucleic acid sequence for the 8.11.3F heavy
chain (SEQ ID NO: 1). FIG. 12B shows the full-length amino acid
sequence for the 8.11.3F heavy chain (SEQ ID NO: 2), and the amino
acid sequence for the 8.11.3F heavy chain amino acid variable
region is in upper case and designated between brackets "[ ]" (SEQ
ID NO: 5). The amino acid sequence of each 8.11.3F heavy chain CDR
is underlined and in lowercase. The heavy chain CDR amino acid
sequences are as follows: CDR1: GFTFSSFSMT (SEQ ID NO: 7); CDR2:
YISSRSSTISYADSVKG (SEQ ID NO: 8); and CDR3: DPLLAGATFFDY (SEQ ID
NO: 9). FIG. 12C shows the nucleic acid sequence for the
full-length 8.11.3F light chain (SEQ ID NO: 3). FIG. 12D shows the
amino acid sequence of the full-length 8.11.3F light chain (SEQ ID
NO: 4), and the 8.11.3F light chain amino acid variable region is
in upper case and designated between brackets "[ ]" (SEQ ID NO: 6).
The amino acid sequence of each light chain CDR is indicated as
follows: CDR1: RASQSVSSSYLA (SEQ ID NO: 10); CDR2: GASSRAT (SEQ ID
NO: 11); and CDR3: QQYGSSPLT (SEQ ID NO: 12).
DETAILED DESCRIPTION OF THE INVENTION
[0053] The methods and techniques of the present invention are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification unless otherwise indicated. See, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates (1992), and Harlow and Lane Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1990). Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of subjects.
DEFINITIONS
[0054] In order to aid the reader in understanding the following
detailed description, the following definitions are provided:
[0055] As used herein, the term "antibody" refers to an intact
antibody or an antigen-binding portion that competes with the
intact antibody for specific binding. See generally, Fundamental
Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).
Antigen-binding portions may be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies. In some embodiments, antigen-binding portions include
Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, and complementarity
determining region (CDR) fragments, single-chain antibodies (scFv),
chimeric antibodies, diabodies and polypeptides that contain at
least a portion of an antibody that is sufficient to confer
specific antigen binding to the polypeptide. From N-terminus to
C-terminus, both the mature light and heavy chain variable domains
comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is in accordance with the
definitions of Kabat, Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987 and
1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), or
Chothia et al., Nature 342:878-883 (1989).
[0056] In some embodiments, the antibody is a single-chain antibody
(scFv) in which a V.sub.L and V.sub.H domains are paired to form a
monovalent molecules via a synthetic linker that enables them to be
made as a single protein chain. (Bird et al., Science 242:423-426
(1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883
(1988).) In some embodiments, the antibodies are diabodies, i.e.,
are bivalent antibodies in which V.sub.H and V.sub.L domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites.
(See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123
(1994)). In some embodiments, one or more CDRs from an antibody of
the invention may be incorporated into a molecule either covalently
or noncovalently to make it an immunoadhesin that specifically
binds to M-CSF. In such embodiments, the CDR(s) may be incorporated
as part of a larger polypeptide chain, may be covalently linked to
another polypeptide chain, or may be incorporated
noncovalently.
[0057] As used herein, an antibody that is referred to by number is
the same as a monoclonal antibody that is obtained from the
hybridoma of the same number. For example, monoclonal antibody
8.10.3F is the same antibody as one obtained from hybridoma
8.10.3F. For example, monoclonal antibody 8.10.3F has the same
heavy and light chain amino acid sequences as one obtained from
hybridoma 8.10.3F. Thus, reference to antibody 8.10.3F includes an
antibody, which has the heavy and light chain amino acid sequences
shown in SEQ ID NOS. 30 and 32, respectively. It also includes an
antibody lacking a terminal lysine on the heavy chain, as this is
normally lost in a proportion of antibodies during manufacture.
[0058] As used herein, an Fd fragment means an antibody fragment
that consists of the V.sub.H and C.sub.H1 domains; an Fv fragment
consists of the V.sub.L and V.sub.H domains of a single arm of an
antibody; and a dAb fragment (Ward et al., Nature 341:544-546
(1989)) consists of a V.sub.H domain.
[0059] As used herein, the term "polypeptide" encompasses native or
artificial proteins, protein fragments and polypeptide analogs of a
protein sequence. A polypeptide may be monomeric or polymeric.
[0060] The terms "or an antigen-binding portion thereof" when used
with the term "antibody" refers to a polypeptide that has an
amino-terminal and/or carboxy-terminal deletion, but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence. In some embodiments,
the antigen-binding portion thereof may be at least 14, at least
20, at least 50, or at least 70, 80, 90, 100, 150 or at least 200
amino acids long.
[0061] As used herein, the terms "is capable of specifically
binding" refers to when an antibody binds to an antigen with a
dissociation constant that is .ltoreq.1 .mu.M, preferably .ltoreq.1
nM and most preferably .ltoreq.10 pM. In certain embodiments, the
K.sub.D is 1 pM to 500 pM. In other embodiments, the K.sub.D is
between 500 pM to 1 .mu.M. In other embodiments, the K.sub.D is
between 1 .mu.M to 100 nM. In other embodiments, the K.sub.D is
between 100 mM to 10 nM.
[0062] As used herein, the term "monoclonal antibody" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts or lacking a
C-terminal lysine. Monoclonal antibodies are highly specific, being
directed against a single antigenic site. Furthermore, in contrast
to conventional (polyclonal) antibody preparations, which 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.
[0063] The term "isolated protein", "isolated polypeptide" or
"isolated antibodies" is a protein, polypeptide or antibody that by
virtue of its origin or source of derivation has one to four of the
following: (1) is not associated with naturally associated
components that accompany it in its native state, (2) is free of
other proteins from the same species, (3) is expressed by a cell
from a different species, or (4) does not occur in nature. Thus, a
polypeptide that is chemically synthesized or synthesized in a
cellular system different from the cell from which it naturally
originates will be "isolated" from its naturally associated
components. An isolated protein/antibody may also be rendered
substantially free of naturally associated cellular components by
isolation, using protein purification techniques well known in the
art.
[0064] Examples of isolated/purified antibodies include an
anti-M-CSF antibody that has been affinity purified using M-CSF, an
anti-M-CSF antibody that has been synthesized by a hybridoma or
other cell line in vitro, and a human anti-M-CSF antibody derived
from a transgenic mouse. Thus, in preferred embodiments, the
anti-M-CSF antibodies have a purity of at least about 95%
(w/w--weight anti-M-CSF antibodies/weight of components other than
pharmaceutically acceptable excipients), and in further
embodiments, the anti-M-CSF antibodies have a purity from about 95%
w/w to about 99.5% w/w.
[0065] An antibody is "substantially pure," "substantially
homogeneous," or "substantially purified" when at least about 60 to
75% of a sample exhibits a single species of antibody. The antibody
may be monomeric or multimeric. A substantially pure antibody can
typically comprise about 50%, 60%, 70%, 80% or 90% w/w of an
antibody sample, more usually about 95%, and preferably will be
over 99% pure. Antibody purity or homogeneity may be indicated by a
number of means well known in the art, such as polyacrylamide gel
electrophoresis of an antibody sample, followed by visualizing a
single polypeptide band upon staining the gel with a stain well
known in the art. For certain purposes, higher resolution may be
achieved by using HPLC or other means well known in the art for
purification.
[0066] As used herein, the term "human antibody" is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0067] As used herein, the term "recombinant human antibody" is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial human antibody library, antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992)
Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,
created or isolated by any other means that involves splicing of
human immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions
derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies are
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the VH and VL regions of the
recombinant antibodies are sequences that, while derived from and
related to human germline VH and VL sequences, may not naturally
exist within the human antibody germline repertoire in vivo.
[0068] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor or
otherwise interacting with a molecule. Epitopic determinants
generally consist of chemically active surface groupings of
molecules such as amino acids or sugar side chains and generally
have specific three-dimensional structural characteristics, as well
as specific charge characteristics. An epitope may be "linear" or
"conformational." In a linear epitope, all of the points of
interaction between the protein and the interacting molecule (such
as an antibody) occur linearly along the primary amino acid
sequence of the protein. In a conformational epitope, the points of
interaction occur across amino acid residues on the protein that
are separated from one another.
[0069] As used herein, the term "polynucleotide" or "nucleic acid",
used interchangeably herein, means a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms. A
"polynucleotide" or a "nucleic acid" sequence encompasses its
complement unless otherwise specified. Thus, a reference to a
nucleic acid having a particular sequence should be understood to
encompass its complementary strand, with its complementary
sequence.
[0070] As used herein, the term "isolated polynucleotide" or
"isolated nucleic acid" means a polynucleotide of genomic, cDNA, or
synthetic origin or some combination thereof, which by virtue of
its origin or source of derivation, the isolated polynucleotide has
one to three of the following: (1) is not associated with all or a
portion of a polynucleotide with which the "isolated
polynucleotide" is found in nature, (2) is operably linked to a
polynucleotide to which it is not linked in nature, or (3) does not
occur in nature as part of a larger sequence.
[0071] The term "oligonucleotide" as used herein includes naturally
occurring, and modified nucleotides linked together by naturally
occurring and non-naturally occurring oligonucleotide linkages.
Oligonucleotides are a polynucleotide subset generally comprising a
length of 200 bases or fewer. Preferably oligonucleotides are 10 to
60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18,
19, or 20 to 40 bases in length. Oligonucleotides are usually
single stranded, e.g. for primers and probes; although
oligonucleotides may be double stranded, e.g. for use in the
construction of a gene mutant. Oligonucleotides of the invention
can be either sense or antisense oligonucleotides.
[0072] As used herein, the term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" as Used herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotides linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the
like. See e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986);
Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl.
Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design
6:539 (1991); Zon et al., Oligonucleotides and Analogues: A
Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University
Press, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann and
Peyman, Chemical Reviews 90:543 (1990), the disclosures of which
are hereby incorporated by reference. An oligonucleotide can
include a label for detection, if desired.
[0073] As used herein, the terms "selectively hybridize" mean to
detectably and specifically bind. Polynucleotides, oligonucleotides
and fragments thereof in accordance with the invention selectively
hybridize to nucleic acid strands under hybridization and wash
conditions that minimize appreciable amounts of detectable binding
to nonspecific nucleic acids. "High stringency" or "highly
stringent" conditions can be used to achieve selective
hybridization conditions as known in the art and discussed herein.
One example of "high stringency" or "highly stringent" conditions
is the incubation of a polynucleotide with another polynucleotide,
wherein one polynucleotide may be affixed to a solid surface such
as a membrane, in a hybridization buffer of 6.times.SSPE or SSC,
50% formamide, 5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml
denatured, fragmented salmon sperm DNA at a hybridization
temperature of 42.degree. C. for 12-16 hours, followed by twice
washing at 55.degree. C. using a wash buffer of 1.times.SSC, 0.5%
SDS. See also Sambrook et al., supra, pp. 9.50-9.55.
[0074] As applied to polynucleotides, the terms "substantial
identity", "percent identity" or "% identical" mean the percent of
residues when a first contiguous sequence is compared and aligned
for maximum correspondence to a second contiguous sequence. The
length of sequence identity comparison may be over a stretch of at
least about nine nucleotides, usually at least about 18
nucleotides, more usually at least about 24 nucleotides, typically
at least about 28 nucleotides, more typically at least about 32
nucleotides, and preferably at least about 36, 48 or more
nucleotides. The terms "substantial identity", "percent identity"
or "% identical" mean that when a polynucleotide molecule is
optimally aligned with appropriate nucleotide insertions or
deletions with another polynucleotide molecule (or its
complementary strand), there is polynucleotide sequence identity of
at least about 85%, preferably at least about 90%, and more
preferably at least about 95%, 96%, 97%, 98% or 99% of the
nucleotide bases, as measured by any well-known algorithm of
sequence identity, such as FASTA, BLAST or Gap there are a number
of different algorithms known in the art that can be used to
measure polynucleotide sequence identity. For instance,
polynucleotide sequences can be compared using FASTA, Gap or
Bestfit, which are programs in Wisconsin Package Version 10.0,
Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes,
e.g., the programs FASTA2 and FASTA3, provides alignments and
percent sequence identity of the regions of the best overlap
between the query and search sequences (Pearson, Methods Enzymol.
183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000);
Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol.
Biol. 276:71-84 (1998)). Unless otherwise specified, default
parameters for a particular program or algorithm are used. For
instance, percent sequence identity between nucleic acid sequences
can be determined using FASTA with its default parameters (a word
size of 6 and the NOPAM factor for the scoring matrix) or using Gap
with its default parameters as provided in GCG Version 6.1, herein
incorporated by reference.
[0075] As applied to polypeptides, the terms "substantial
identity", "percent identity" or "% identical" mean that two
peptide sequences, when optimally aligned, such as by the programs
GAP or BESTFIT using default gap weights, as supplied with the
programs, share at least 70%, 75% or 80% sequence identity,
preferably at least 90% or 95% sequence identity, and more
preferably at least 97%, 98% or 99% sequence identity. In certain
embodiments, residue positions that are not identical differ by
conservative amino acid substitutions. A "conservative amino acid
substitution" is one in which an amino acid residue is substituted
by another amino acid residue having a side chain R group with
similar chemical properties (e.g., charge or hydrophobicity). In
general, a conservative amino acid substitution will not
substantially change the functional properties of a protein. In
cases where two or more amino acid sequences differ from each other
by conservative substitutions, the percent sequence identity may be
adjusted upwards to correct for the conservative nature of the
substitution. Means for making this adjustment are well-known to
those of skill in the art. See, e.g., Pearson, Methods Mol. Biol.
243:307-31 (1994). Examples of groups of amino acids that have side
chains with similar chemical properties include 1) aliphatic side
chains: glycine, alanine, valine, leucine, and isoleucine; 2)
aliphatic-hydroxyl side chains: serine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; 6) acidic side chains:
aspartic acid and glutamic acid; and 7) sulfur-containing side
chains: cysteine and methionine. Conservative amino acids
substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine. Sequence identity
for polypeptides, is typically measured using sequence analysis
software. Protein analysis software matches sequences using
measures of similarity assigned to various substitutions, deletions
and other modifications, including conservative amino acid
substitutions. For instance, GCG contains programs such as "Gap"
and "Bestfit" which can be used with default parameters, as
specified with the programs, to determine sequence homology or
sequence identity between closely related polypeptides, such as
homologous polypeptides from different species of organisms or
between a wild type protein and a mutant thereof. See, e.g., GCG
Version 6.1. Polypeptide sequences also can be compared using FASTA
using default or recommended parameters, see GCG Version 6.1.
(University of Wisconsin WI) FASTA (e.g., FASTA2 and FASTA3)
provides alignments and percent sequence identity of the regions of
the best overlap between the query and search sequences (Pearson,
Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.
32:185-219 (2000)). Another preferred algorithm when comparing a
sequence of the invention to a database containing a large number
of sequences from different organisms is the computer program
BLAST, especially blastp or tblastn, using default parameters, as
supplied with the programs. See, e.g., Altschul et al., J. Mol.
Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.
25:3389-402 (1997). The length of polypeptide sequences compared
for homology will generally be at least about 16 amino acid
residues, usually at least about 20 residues, more usually at least
about 24 residues, typically at least about 28 residues, and
preferably more than about 35 residues. When searching a database
containing sequences from a large number of different organisms, it
is preferable to compare amino acid sequences.
[0076] "Operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest. The term "expression control
sequence" as used herein means polynucleotide sequences that are
necessary to effect the expression and processing of coding
sequences to which they are ligated. Expression control sequences
include appropriate transcription initiation, termination, promoter
and enhancer sequences; efficient RNA processing signals such as
splicing and polyadenylation signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(i.e., Kozak consensus sequence); sequences that enhance protein
stability; and when desired, sequences that enhance protein
secretion. The nature of such control sequences differs depending
upon the host organism; in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence; in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression
and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences.
[0077] As used herein, the term "vector" means a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. In some embodiments, the vector is a plasmid,
i.e., a circular double stranded DNA loop into which additional DNA
segments may be ligated. In some embodiments, the vector is a viral
vector, wherein additional DNA segments may be ligated into the
viral genome. In some embodiments, the vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). In other embodiments,
the vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors").
[0078] As used herein, the terms "recombinant host cell" (or simply
"host cell") means a cell into which a recombinant expression
vector has been introduced. It should be understood that
"recombinant host cell" and "host cell" mean not only the
particular subject cell but also the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used
herein.
[0079] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, which includes treatment or
prophylactic prevention of any M-CSF meditated condition, including
inflammatory diseases and neoplasia disorders. It is to be noted
that dosage values may vary with the severity of the condition to
be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition. Likewise, a
therapeutically effective amount of the antibody or antibody
portion may vary according to factors such as the disease state,
age, sex, and weight of the individual, the ability of the antibody
or antibody portion to elicit a desired response in the individual,
and the desired route of administration of the antibody
formulation. A therapeutically effective amount is also one in
which any toxic or detrimental effects of the antibody or antibody
portion are outweighed by the therapeutically beneficial
effects.
[0080] As used herein, the term "subject" for purposes of treatment
includes any subject, and preferably is a subject who is in need of
the treatment of an M-CSF-mediated disorder. For purposes of
prevention, the subject is any subject, and preferably is a subject
that is at risk for, or is predisposed to, developing an
M-CSF-mediated disorder. The term "subject" is intended to include
living organisms, e.g., prokaryotes and eukaryotes. Examples of
subjects include mammals, e.g., humans, dogs, cows, horses, pigs,
sheep, goats, cats, mice, rabbits, rats, and transgenic non-human
animals. In specific embodiments of the invention, the subject is a
human.
[0081] As used herein, the term "M-CSF-mediated disorder" is
intended to include diseases and other disorders in which the
presence of M-CSF in a subject suffering from the disorder is
elevated in comparison to a normal healthy subject, whether the
elevated M-CSF levels are now known or later evidenced or suspected
of being either responsible for the pathophysiology of the disorder
or a factor that contributes to a worsening of the disorder. Such
disorders may be evidenced, for example, by an increase in the
levels of M-CSF secreted and/or on the cell surface or increased
tyrosine autophosphorylation of c-fms in the affected cells or
tissues of a subject suffering from the disorder. The increase in
M-CSF levels may be detected, for example, using an anti-M-CSF
antibody as would be understood by one of skill in the art.
Examples of M-CSF-mediated disorders that are encompassed by the
present invention include inflammatory diseases, cardiovascular
disorders, and neoplasia disorders.
[0082] As used herein, the terms "neoplasia" and "neoplasia
disorders", used interchangeably herein, refer to new cell growth
that results from a loss of responsiveness to normal growth
controls, e.g. to "neoplastic" cell growth. Neoplasia is also used
interchangeably herein with the term "cancer" and for purposes of
the present invention; cancer is one subtype of neoplasia. As used
herein, the term "neoplasia disorder" also encompasses other
cellular abnormalities, such as hyperplasia, metaplasia and
dysplasia. The terms neoplasia, metaplasia, dysplasia and
hyperplasia can be used interchangeably herein and refer generally
to cells experiencing abnormal cell growth.
[0083] As used herein, the term "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or condition. Those in need of treatment
include those already with the condition as well as those prone to
have the condition or those in whom the condition is to be
prevented.
[0084] When introducing elements of the present invention or the
preferred embodiment(s) thereof, the articles "a", "an", "the" and
"said" are intended to mean that there are one or more of the
elements. Throughout this specification and claims, the terms
"comprising", "comprise", "comprises", "including" and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
Anti-M-CSF Antibodies:
[0085] In accordance with the present invention, it has been
discovered that the stability of certain monoclonal anti-M-CSF
antibodies that are described herein can be improved by mixing the
anti-M-CSF antibodies with a chelating agent, such as
ethylenediaminetetraacetic acid ("EDTA").
[0086] While not wishing to be bound by theory, it is believed that
the presence of a chelating agent in the compositions of the
present invention help to improve stability of the antibody
polypeptide by reducing the incidence of one or more of the
following: anti-M-CSF antibody aggregation, fragmentation,
oxidation, freeze/thaw instability, discoloration, and/or
deamidation. The present invention comprises anti-M-CSF antibody
compositions having improved chemical and/or physical stability as
compared to previously disclosed antibody compositions.
[0087] Therefore, in certain aspects, the present invention
provides at least one composition comprising a chelating agent,
such as EDTA and at least one monoclonal anti-M-CSF antibody or an
antigen-binding portion thereof. In still other aspects, the
aforementioned anti-M-CSF antibody compositions comprising a
chelating agent can include additional excipients, including, but
not limited to, one or more excipients that are chosen from
buffers, tonicity agents, surfactants, antioxidants, and mixtures
thereof.
[0088] The present invention provides novel formulations for
anti-M-CSF antibodies. As used herein, the phrase "anti-M-CSF
antibody" refers to any antibody, or any portion thereof, that is
capable of binding to any portion of a macrophage
colony-stimulating factor ("M-CSF") polypeptide that may be present
within or isolated from any animal. In certain embodiments, the
M-CSF polypeptide is a human M-CSF polypeptide.
[0089] Suitable anti-M-CSF antibodies for use with the present
invention may be chosen from polyclonal or monoclonal antibodies.
In certain aspects, the monoclonal anti-M-CSF antibody can be a
murine, chimeric, humanized or human antibody. In further
embodiments, the monoclonal anti-M-CSF antibody is a human
monoclonal anti-M-CSF antibody. In further embodiments, the
monoclonal anti-M-CSF antibody is an isolated monoclonal anti-M-CSF
antibody. In further embodiments, the monoclonal anti-M-CSF
antibody is a recombinant monoclonal anti-M-CSF antibody.
[0090] In certain embodiments, the anti-M-CSF antibodies which are
suitable for use with the present invention include those
anti-M-CSF antibodies and methods to prepare them that are
described in U.S. Published Application No. 20050059113 to Bedian,
et al. In other embodiments, the anti-M-CSF antibodies which are
suitable for use with the present invention include any one or more
of those anti-M-CSF monoclonal antibodies having the heavy and
light chain amino acid sequences of the antibodies designated 252,
88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.41, 9.7.21F, 9.14.4, 8.10.3,
9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2,
9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser,
8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1 in U.S. Published
Application No. 20050059113 to Bedian, et al. In still other
embodiments, the anti-M-CSF antibodies which are suitable for use
with the present invention include those anti-M-CSF monoclonal
antibodies having the heavy and light chain amino acid sequences of
the antibody designated 8.10.3F in U.S. Published Application No.
20050059113 to Bedian, et al.
[0091] In addition, such anti-M-CSF antibodies may be chosen based
on differences in the amino acid sequences in the constant region
of their heavy chains. For example, the anti-M-CSF antibodies may
be chosen from the IgG class, which have "gamma" type heavy chains.
The class and subclass of anti-M-CSF antibodies may be determined
by any method known in the art. In general, the class and subclass
of an antibody may be determined using antibodies that are specific
for a particular class and subclass of antibody. Such antibodies
are commercially available. The class and subclass can be
determined by ELISA, or Western Blot as well as other techniques.
Alternatively, the class and subclass may be determined by
sequencing all or a portion of the constant domains of the heavy
and/or light chains of the antibodies, comparing their amino acid
sequences to the known amino acid sequences of various class and
subclasses of immunoglobulins, and determining the class and
subclass of the antibodies.
[0092] The anti-M-CSF antibody can be an IgG, an IgM, an IgE, an
IgA, or an IgD molecule. In further embodiments, the anti-M-CSF
antibody is an IgG and is an IgG1, IgG2, IgG3 or IgG4 subclass. One
of the major mechanisms through which antibodies kill cells is
through fixation of complement and participation in CDC. The
constant region of an antibody plays an important role in
connection with an antibody's ability to fix complement and
participate in CDC. Thus, generally one selects the isotype of an
antibody to either provide the ability of complement fixation, or
not. In the case of the present invention, generally, as mentioned
above, it is generally not preferred to utilize an antibody that
kills the cells. There are a number of isotypes of antibodies that
are capable of complement fixation and CDC, including, without
limitation, the following: murine IgM, murine IgG2a, murine IgG2b,
murine IgG3, human IgM, human IgG1, and human IgG3. In contrast,
preferred isotypes which are not capable of complement fixation and
CDC include, without limitation, human IgG2 and human IgG4. In
addition to heavy chain sequence differences, the IgG antibodies
differ within their subclass based on the number of disulfide bonds
and length of the hinge region. For example, the IgG2 subclass has
several differences distinct from the other subclasses. The IgG2
and IgG4 subclasses are known to have 4 disulfide bonds within
their hinge region, while IgG1 has 2 and IgG3 has 11 disulfide
bonds. Other differences for IgG2 antibodies include their reduced
ability to cross the placenta and the inability of IgG2 antibodies
to bind to lymphocyte Fc receptors. Thus, in certain embodiments,
the anti-M-CSF antibody is subclass IgG2 or IgG4. In another
preferred embodiment, the anti-M-CSF antibody is subclass IgG2.
[0093] In other embodiments, suitable anti-M-CSF antibodies may be
chosen based on differences in the amino acid sequences in their
heavy chains. For example, the anti-M-CSF antibodies of the present
invention may have human gamma type heavy chains that utilize any
of the following human V.sub.H germline genes: V.sub.H1, V.sub.H2,
V.sub.H3, V.sub.H4, or V.sub.H5. For purposes of the present
invention, the phrase "heavy chain variable regions" is often
abbreviated with the term (V.sub.H). In certain embodiments, the
anti-M-CSF antibodies utilize the human V.sub.H3 germline gene. In
further embodiments, the anti-M-CSF antibodies utilize the human
V.sub.H3-48 germline gene. In still further embodiments, the
anti-M-CSF antibodies utilize the D1-26 human D.sub.H gene. In
still further embodiments, the anti-M-CSF antibodies utilize the
J.sub.H4 human J.sub.H gene.
[0094] In further embodiments, the anti-M-CSF antibodies may be
chosen based on differences in the amino acid sequences of their
light chains. For example, suitable anti-M-CSF antibodies may have
lambda light chains or kappa light chains. However, in certain
embodiments, the anti-M-CSF antibodies of the present invention
have kappa light chains. In some embodiments, where the anti-M-CSF
antibody comprises a kappa light chain, the polynucleotide encoding
the variable domain of the light chain comprises a human V.sub.K
L5, O12, L2, B3, L15, or A27 gene and a human Jk1, Jk2, Jk3, Jk4,
or Jk5 gene. In some embodiments where the antibody comprises a
kappa light chain, the light chain variable region (V.sub.L) is
encoded in part by a human V.sub.KA27 gene and a human J.sub.K4
gene. In particular embodiments of the invention, the light chain
variable domain is encoded by human V.sub.KA27/Jk3 genes.
[0095] Table 1 lists the sequence identifiers (SEQ ID NOS) of the
nucleic acids that comprise the heavy and light chains and the
corresponding predicted amino acid sequences for the anti-M-CSF
monoclonal antibody 8.10.3F. While DNA sequences encoding a signal
polypeptide are shown in the sequence identifiers (SEQ ID NOS), the
antibody typically does not comprise a signal polypeptide because
the signal polypeptide is generally eliminated during
post-translational modifications. In various embodiments of the
invention, one or both of the heavy and light chains of the
anti-M-CSF antibodies includes a signal sequence (or a portion of
the signal sequence). In other embodiments of the invention,
neither the heavy nor light chain of the anti-M-CSF antibodies
includes a signal sequence. Table 1 also lists the heavy chain and
light chain human germline gene derivation and sequences for the
anti-M-CSF monoclonal antibody 8.10.3F.
TABLE-US-00001 TABLE 1 Heavy and Light Chain Human Gene Utilization
and Sequences Heavy Chain Light Chain Antibody SEQ ID NO: V.sub.H
D.sub.H J.sub.H SEQ ID NO: V.sub.K J.sub.K 8.10.3F 1 (nucleic 3-48
1-26 4b 3 (nucleic A27 4 acid) acid) 2 (amino 4 (amino acid)
acid)
[0096] Some anti-M-CSF antibodies in accordance with the present
invention were generated with a bias towards the utilization of the
human V.sub.H3-48 heavy chain variable region. In XenoMouse.TM.
mice, there are more than 30 distinct functional heavy chain
variable genes with which to generate antibodies. Bias, therefore,
is indicative of a preferred binding motif of the antibody-antigen
interaction with respect to the combined properties of binding to
the antigen and functional activity.
[0097] In some embodiments, the nucleic acid molecule encodes an
amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17 or 18 mutations compared to the germline
amino acid sequence of the human V, D or J genes. In some
embodiments, said mutations are in the heavy chain variable region
region. In some embodiments, said mutations are in the CDR
regions.
[0098] In some embodiments, the nucleic acid molecule encodes one
or more amino acid mutations compared to the germline sequence that
are identical to amino acid mutations found in the V.sub.H of
monoclonal antibody 8.10.3F. In some embodiments, the nucleic acid
encodes at least three amino acid mutations compared to the
germline sequences that are identical to at least three amino acid
mutations found in one of the above-listed monoclonal
antibodies.
[0099] In some embodiments, the nucleic acid molecule encodes a
V.sub.L amino acid sequence comprising one or more variants
compared to germline sequence that are identical to the variations
found in the V.sub.L of one of the antibodies 8.10.3F.
[0100] In some embodiments, the nucleic acid molecule encodes at
least three amino acid mutations compared to the germline sequence
found in the V.sub.L of the antibody 8.10.3.
[0101] In some embodiments, the antibody is a single-chain antibody
(scFv) in which a V.sub.L and V.sub.H domains are paired to form a
monovalent molecules via a synthetic linker that enables them to be
made as a single protein chain. Bird et al., Science 242:423-426
(1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883
(1988). In some embodiments, the antibodies are diabodies, i.e.,
are bivalent antibodies in which V.sub.H and V.sub.L domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites.
See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993 and Poljak R. J. et al., Structure 2:1121-1123
(1994). In some embodiments, one or more CDRs from an antibody of
the invention may be incorporated into a molecule either covalently
or noncovalently to make it an immunoadhesin that specifically
binds to M-CSF. In such embodiments, the CDR(s) may be incorporated
as part of a larger polypeptide chain, may be covalently linked to
another polypeptide chain, or may be incorporated
noncovalently.
[0102] In another embodiment, the anti-M-CSF antibody has
selectivity (or specificity) for M-CSF that is at least 100 times
greater than its selectivity for any other polypeptide. In some
embodiments, the anti-M-CSF antibody does not exhibit any
appreciable specific binding to any other protein other than M-CSF.
One can determine the selectivity of the anti-M-CSF antibody for
M-CSF using methods well known in the art following the teachings
of the specification. For instance, one can determine the
selectivity using Western blot, FACS, ELISA, or RIA. Thus, in some
embodiments, the monoclonal anti-M-CSF antibody is capable of
specifically binding to M-CSF.
[0103] In some embodiments, the C-terminal lysine of the heavy
chain of the anti-M-CSF antibody of the invention is not
present.
[0104] In some embodiments, the nucleic acid molecule encodes a
light chain amino acid sequence that is at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a light chain
amino acid sequence of antibody 8.10.3F of SEQ ID NO: 4, or to a
V.sub.L amino acid sequence of SEQ ID NO 6. Nucleic acid molecules
of the invention include nucleic acids that hybridize under highly
stringent conditions, such as those described above, to a nucleic
acid sequence encoding the light chain amino acid sequence of SEQ
ID NO: 4, or that has the polynucleotide sequence of SEQ ID NO:
3.
[0105] In some embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes the light chain amino acid
sequence of monoclonal antibody 8.10.3F, or a portion thereof. In
some embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes the light chain polynucleotide
sequence of monoclonal antibody 8.10.3F of SEQ ID NO: 3, or a
portion thereof. In some embodiments, the nucleic acid molecule
comprises a polynucleotide sequence that encodes the V.sub.L amino
acid sequence of monoclonal antibody 8.1.0.3F of SEQ ID NO: 6, or a
portion thereof. In some embodiments, said portion comprises at
least the CDR2 region. In some embodiments, the nucleic acid
encodes the amino acid sequence of the light chain CDRs of said
antibody. In some embodiments, said portion is a contiguous portion
comprising CDR1-CDR3.
[0106] In some embodiments, the nucleic acid molecule encodes a
heavy chain amino acid sequence that is at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a heavy chain
amino acid sequence of antibody 8.10.3F of SEQ ID NO: 2, or to a
V.sub.H amino acid sequence of SEQ ID NO 5. Nucleic acid molecules
of the invention include nucleic acids that hybridize under highly
stringent conditions, such as those described above, to a nucleic
acid sequence encoding the heavy chain amino acid sequence of SEQ
ID NO: 2, or that has the polynucleotide sequence of SEQ ID NO:
1.
[0107] In some embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes the heavy chain amino acid
sequence of monoclonal antibody 8.10.3F, or a portion thereof. In
some embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes the heavy chain polynucleotide
sequence of monoclonal antibody 8.10.3F of SEQ ID NO: 2, or a
portion thereof. In some embodiments, the nucleic acid molecule
comprises a polynucleotide sequence that encodes the V.sub.H amino
acid sequence of monoclonal antibody 8.10.3F of SEQ ID NO: 5, or a
portion thereof. In some embodiments, said portion comprises at
least the CDR2 region. In some embodiments, the nucleic acid
encodes the amino acid sequence of the light chain CDRs of said
antibody. In some embodiments, said portion is a contiguous portion
comprising CDR1-CDR3.
[0108] In further embodiments, the nucleic acid molecule comprises
a polynucleotide sequence that encodes at least a portion of the
V.sub.H amino acid sequence of 8.10.3F (SEQ ID NO: 5) or said
sequence having conservative amino acid mutations and/or a total of
three or fewer non-conservative amino acid substitutions. In
various embodiments the sequence encodes one or more CDR regions,
preferably a CDR3 region, all three CDR regions, a contiguous
portion including CDR1-CDR3, or the entire V.sub.H region.
[0109] In still further embodiments, the nucleic acid molecule
comprises a polynucleotide sequence that encodes the heavy chain
amino acid sequence of SEQ ID NO: 1 or a portion thereof. In still
further embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes the heavy chain variable
domain amino acid sequence of SEQ ID NO: 5 or a portion
thereof.
[0110] In another embodiment, the nucleic acid encodes a
full-length light chain of an antibody selected from 8.10.3F, or a
light chain comprising the amino acid sequence of SEQ ID NO: 4 and
a constant region of a light chain, or a light chain comprising a
mutation. Further, the nucleic acid may comprise the light chain
polynucleotide sequence of SEQ ID NO: 3 and the polynucleotide
sequence encoding a constant region of a light chain, or a nucleic
acid molecule encoding a light chain comprise a mutation.
[0111] In some embodiments, the nucleic acid molecule comprises a
polynucleotide sequence that encodes at least a portion of the
V.sub.H amino acid sequence of 8.10.3F (SEQ ID NO: 5) or said
sequence having conservative amino acid mutations and/or a total of
three or fewer non-conservative amino acid substitutions. In
various embodiments the sequence encodes one or more CDR regions,
preferably a CDR3 region, all three CDR regions, a contiguous
portion including CDR1-CDR3, or the entire V.sub.H region.
[0112] In another aspect of the invention, the anti-M-CSF
antibodies demonstrate both species and molecule selectivity. In
some embodiments, the anti-M-CSF antibody binds to human,
cynomologus monkey and mouse M-CSF. Following the teachings of the
specification, one may determine the species selectivity for the
anti-M-CSF antibody using methods well known in the art. For
instance, one may determine the species selectivity using Western
blot, FACS, ELISA, RIA, a cell proliferation assay, or an M-CSF
receptor-binding assay. In a preferred embodiment, one may
determine the species selectivity using a cell proliferation assay
or ELISA.
[0113] In another embodiment, the anti-M-CSF antibody has
selectivity for M-CSF that is at least 100 times greater than its
selectivity for GM-/G-CSF. In some embodiments, the anti-M-CSF
antibody does not exhibit any appreciable specific binding to any
other protein other than M-CSF. One can determine the selectivity
of the anti-M-CSF antibody for M-CSF using methods well known in
the art following the teachings of the specification. For instance
one can determine the selectivity using Western blot, FACS, ELISA,
or RIA.
Preparation of the Monoclonal Anti-M-CSF Antibody Formulations:
[0114] The anti-M-CSF antibody typically is formulated as a
pharmaceutical composition for parenteral administration to a
subject. In certain embodiments, the pharmaceutical composition is
a liquid composition.
[0115] In one embodiment, the invention is directed to compositions
comprising an anti-M-CSF antibody and a chelating agent. In another
embodiment, the invention is directed to a liquid pharmaceutical
composition comprising an anti-M-CSF antibody and a chelating
agent. In another embodiment, the invention is directed to a
composition comprising an anti-M-CSF antibody and EDTA. In another
embodiment, the invention is directed to a liquid pharmaceutical
composition comprising an anti-M-CSF antibody and EDTA.
[0116] The term "pharmaceutical composition" refers to preparations
which are in such form as to permit the biological activity of the
active ingredients to be effective. In some embodiments, the
pharmaceutical composition is a liquid pharmaceutical composition.
"Pharmaceutically acceptable excipients" (vehicles, additives) are
those, which can reasonably (i.e., safely) be administered to a
subject to provide an effective dose of the active ingredient
employed. The term "excipient" or "carrier" as used herein refers
to an inert substance, which is commonly used as a diluent,
vehicle, preservative, binder or stabilizing agent for drugs. As
used herein, the term "diluent" refers to a pharmaceutically
acceptable (safe and non-toxic for administration to a human)
solvent and is useful for the preparation of the compositions
herein. Exemplary diluents include, but are not limited to, sterile
water and bacteriostatic water for injection (BWFI).
[0117] The compositions of the present invention involve one or
more anti-M-CSF monoclonal antibodies of the invention in
combination with pharmaceutically acceptable excipients, which
comprise a chelating agent. The compositions of the present
invention involve one or more anti-M-CSF monoclonal antibodies of
the invention in combination with pharmaceutically acceptable
excipients, which comprise histidine and/or a chelating agent.
[0118] In one embodiment, the composition comprises at least one
antibody comprising an amino acid sequence that is at least 90%,
95% or 99% identical to a light chain sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90%, 95%, or 99% identical to a heavy chain amino acid sequence
shown in SEQ ID NO: 2; and a chelating agent, wherein the antibody
binds to human M-CSF.
[0119] In one embodiment, the composition comprises at least one
antibody comprising an amino acid sequence that is at least 90%,
95% or 99% identical to a light chain sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90%, 95%, or 99% identical to a heavy chain amino acid sequence
shown in SEQ ID NO: 2; and histidine, wherein the antibody binds to
human M-CSF.
[0120] In another embodiment, the composition comprises at least
one antibody comprising an amino acid sequence that is at least
90%, 95% or 99% identical to a light chain sequence shown in SEQ ID
NO: 4, and further comprising an amino acid sequence that is at
least 90%, 95%, or 99% identical to a heavy chain amino acid
sequence shown in SEQ ID NO: 2; and a chelating agent, wherein the
antibody binds to human M-CSF and further comprising at least one
or more pharmaceutically acceptable excipient that is chosen from
buffers, tonicity agents, antioxidants, and surfactants.
[0121] In another embodiment, the composition comprises at least
one antibody comprising an amino acid sequence that is at least
90%, 95% or 99% identical to a light chain sequence shown in SEQ ID
NO: 4, and further comprising an amino acid sequence that is at
least 90%, 95%, or 99% identical to a heavy chain amino acid
sequence shown in SEQ ID NO: 2; and histidine, wherein the antibody
binds to human M-CSF and further comprising at least one or more
pharmaceutically acceptable excipient that is chosen from buffers,
chelating agents, tonicity agents, antioxidants, and
surfactants.
[0122] In another embodiment, the composition comprises at least
one antibody comprising a heavy chain amino acid sequence that
comprises the variable region of SEQ ID NO: 2 and a light chain
amino acid sequence that comprises the variable region SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF.
[0123] In another embodiment, the composition comprises at least
one antibody comprising a human monoclonal IgG2 antibody having the
heavy and light chain amino acid sequences of antibody 8.10.3F; and
a chelating agent, wherein the antibody binds to human M-CSF.
[0124] The concentration of the anti-M-CSF antibody in the
compositions of the present invention is generally at least about
0.1 milligram per milliliter (mg/ml) or higher, at least about 1.0
mg/ml or higher, at least about 10 mg/ml or higher, at least about
20 mg/ml or higher, at least about 50 mg/ml or higher, at least
about 100 mg/ml or higher, or at least about 200 mg/ml or higher.
In certain embodiments, the concentration of the anti-M-CSF
antibody generally ranges from about 0.1 mg/ml to about 200 mg/ml,
from about 0.5 mg/ml to about 100 mg/ml, from about 1 mg/ml to
about 50 mg/ml, from about 2.0 mg/ml to about 35 mg/ml, from about
5.0 mg/ml to about 25 mg/ml, or from about 7 mg/ml to about 15
mg/ml. In one embodiment, the concentration of the anti-M-CSF
antibody in the compositions of the present invention is generally
about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, about 50 mg/ml,
about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml,
about 85 mg/ml, or about 100 mg/ml. In another embodiment, the
concentration of the anti-M-CSF antibody in the compositions range
from about 1 mg/ml to about 50 mg/ml. In one embodiment, the
concentration of the anti-M-CSF antibody in the composition is
about 10 mg/ml. In another embodiment, the concentration of the
anti-M-CSF antibody in the composition is about 75 mg/ml.
[0125] In another embodiment, the concentration of the anti-M-CSF
antibody in the liquid pharmaceutical composition ranges from about
50 mg/ml to about 100 mg/ml. In some embodiments, higher antibody
concentrations can be used where the composition is intended for
subcutaneous delivery.
[0126] As used herein, the terms "chelating agent" generally refers
to an excipient that can form at least one bond (e.g., covalent,
ionic, or otherwise) to a metal ion. A chelating agent is typically
a multidentate ligand that can be used in selected liquid
compositions as a stabilizer to complex with species, which might
promote instability. Often, compounds that can act as a chelating
agent will have electron-rich functional groups. Suitable
electron-rich functional groups include carboxylic acid groups,
hydroxy groups and amino groups. Arrangement of these groups in
aminopolycarboxylic acids, hydroxypolycarboxylic acids,
hydroxyaminocarboxylic acids, and the like, result in moieties that
have the capacity to bind metal.
[0127] However, the present invention is not intended to be limited
to chelating agents primarily by the chelating agent's ability to
form bonds with a metal ion. Therefore, the present invention is
not intended to be limited by any specific mechanism by which the
chelating agent acts in the formulations of the present invention
and the excipients termed chelating agents herein may achieve their
properties through mechanisms that are altogether unrelated to the
chelating agent's ability to form bonds with a metal ion.
[0128] Chelating agents that are suitable for use in the present
invention, include, but are not limited to, aminopolycarboxylic
acids, hydroxyaminocarboxylic acids, N-substituted glycines,
2-(2-amino-2-oxoethyl)aminoethane sulfonic acid (BES), deferoxamine
(DEF), citric acid, niacinamide, and desoxycholates. Examples of
suitable aminopolycarboxylic acids include
ethylenediaminetetraacetic acid (EDTA), diethylenetriamine
pentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA),
N-2-acetamido-2-iminodiacetic acid (ADA),
bis(aminoethyl)glycolether, N,N,N',N'-tetraacetic acid (EGTA),
trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid,
and aspartic acid. Examples of suitable hydroxyaminocarboxylic
acids include N-hydroxyethyliminodiacetic acid (HIMDA),
N,N-bis-hydroxyethylglycine (bicine) and
N-(trishydroxymethylmethyl) 10 glycine (tricine). An example of a
suitable N-substituted glycine is glycylglycine. An example of a
suitable desoxycholate is sodium desoxycholate. Mixtures of two or
more chelating agents are also encompassed by the present
invention.
[0129] Chelating agents used in the invention can be present, where
possible, as the free acid or free base form of the compound (e.g.,
referred to interchangeably herein as "EDTA" or "edetate") or as a
corresponding salt form (e.g., the corresponding acid addition salt
or base addition salt, such as disodium edetate). Suitable acid
addition salts, e.g., include alkali metal salts (e.g., sodium or
potassium salts), alkaline earth metal salts (e.g., calcium salts),
and salts can be prepared using other weakly bound metal ions. As
is known in the art, the nature of the salt and the number of
charges to be neutralized will depend on the number of carboxyl
groups present and the pH at which the stabilizing chelating agent
is supplied. As is also known in the art, chelating agents have
varying strengths with which particular target ions are bound. By
way of further illustration, suitable salts of EDTA include
dipotassium edetate, disodium edetate, edetate calcium disodium,
sodium edetate, trisodium edetate, and potassium edetate; and a
suitable salt of deferoxamine (DEF) is deferoxamine mesylate
(DFM).
[0130] Chelating agents used in the invention can be present as an
anhydrous, solvated or hydrated form of the compound or
corresponding salt. Where the chelating agent is in a solvated or
hydrated form, it can be present in varying states of solvation or
hydration (including, e.g., anhydrous, hydrated, dihydrated, and
trihydrated forms). By way of further illustration, a suitable
hydrate of EDTA is disodium EDTA dihydrate; and suitable forms of
citric acid include anhydrous citric acid, citric acid monohydrate,
and trisodium citrate-dihydrate.
[0131] Suitable chelating agents used in the antibody compositions
of the present invention also include, for example, those that bind
to metal ions in solution to render them unable to react with
available O.sub.2, thereby minimizing or preventing generation of
hydroxyl radicals which are free to react with and degrade the
antibody. Chelating agents can lower the formation of reduced
oxygen species, reduce acidic species (e.g., deamidation)
formation, reduce antibody aggregation, and/or reduce antibody
fragmentation in the compositions of the present invention. Such
chelating agents can reduce or prevent degradation of an antibody
that is formulated without the protection of a chelating agent.
[0132] When a concentration of a chelating agent is referred to, it
is intended that the recited concentration represent the molar
concentration of the free acid or free base form of the chelating
agent. For example, the concentration of chelating agent in certain
liquid pharmaceutical compositions generally ranges from about 0.01
micromolar to about 50 millimolar, from about 1 micromolar to about
10.0 millimolar, from about 15 micromolar to about 5.0 millimolar,
from about 0.01 millimolar to about 1.0 millimolar, or from about
0.03 millimolar to about 0.5 millimolar. In certain embodiments,
the concentration of chelating agent in the liquid pharmaceutical
composition can be about 0.01 millimolar, 0.02 millimolar, 0.027
millimolar, 0.03 millimolar, about 0.04 millimolar, about 0.05
millimolar, about 0.06 millimolar, about 0.07 millimolar, about
0.10 millimolar, about 0.20 millimolar, about 0.26 millimolar,
about 0.27 millimolar, about 0.30 millimolar, about 0.31
millimolar, about 0.34 millimolar, about 0.40 millimolar, about
0.50 millimolar, or about 1.0 millimolar. In certain embodiments,
the concentration of chelating agent is about 0.027 millimolar,
about 0.05 millimolar, about 0.13 millimolar, or about 0.27
millimolar. In one embodiment, the concentration of chelating agent
is about 0.05 millimolar. In another embodiment, the concentration
of chelating agent is about 0.13 millimolar.
[0133] Unless stated otherwise, the concentrations listed herein
are those concentrations at ambient conditions, (i.e., at
25.degree. C. and atmospheric pressure). Ranges intermediate to the
above-recited chelating agent concentrations are also intended to
be part of this invention. For example, ranges of values using a
combination of any of the above-recited values as upper and/or
lower limits are intended to be included.
[0134] In one embodiment, the chelating agent is selected from the
group consisting of EDTA, DTPA, DFM, and mixtures thereof. In
another embodiment, the chelating is agent is DFM. In another
embodiment, the chelating agent is EDTA. In another embodiment, the
chelating agent is DTPA. In another embodiment, the liquid
pharmaceutical composition comprises EDTA in an amount that
generally ranges from about 0.01 micromolar to about 50 millimolar,
from about 1 micromolar to about 20.0 millimolar, from about 15
micromolar to about 10.0 millimolar, from about 0.01 millimolar to
about 5.0 millimolar, or from about 0.03 millimolar to about 1
millimolar. In certain embodiments, the concentration of EDTA in
the liquid pharmaceutical composition can be about 0.01 millimolar,
0.02 millimolar, 0.027 millimolar, 0.03 millimolar, about 0.04
millimolar, about 0.05 millimolar, about 0.06 millimolar, about
0.07 millimolar, about 0.10 millimolar, about 0.20 millimolar,
about 0.26 millimolar, about 0.27 millimolar, about 0.30
millimolar, about 0.31 millimolar, about 0.34 millimolar, about
0.40 millimolar, about 0.50 millimolar, or about 1.0 millimolar. In
certain embodiments, the concentration of EDTA is about 0.027
millimolar, about 0:05 millimolar, about 0.13 millimolar, or about
0.27 millimolar. In one embodiment, the concentration of EDTA is
about 0.05 millimolar. In another embodiment, the concentration of
EDTA is about 0.13 millimolar.
[0135] As noted above, the compositions of the present invention
optionally may further comprise a buffer in addition to a chelating
agent. As used herein, the term "buffer" refers to an added
composition that allows a liquid antibody formulation to resist
changes in pH.
[0136] In certain embodiments, the added buffer allows a liquid
antibody formulation to resist changes in pH by the action of its
acid-base conjugate components. For example, a buffered formulation
may be prepared by adding L-histidine-HCl
(L-histidine-hydrochloride) and L-histidine in the appropriate
amounts to arrive at a desired pH. However, in other embodiments,
the added buffer allows a liquid antibody formulation to resist
changes in pH by the action of its acid-base conjugate components.
By way of a second example, a buffered formulation may be prepared
by adding an acid, such as hydrochloric acid, and L-histidine in
the appropriate amounts to arrive at a desired pH.
[0137] Examples of suitable buffers include, but are not limited
to, acetate (e.g., sodium acetate), succinate (e.g., sodium
succinate), gluconate, citrate (e.g., and other organic acid
buffers, including, but not limited to, buffers such as amino acids
(e.g., histidine), acetic acid, phosphoric acid and phosphates,
ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic
acid, ascorbic acid, imidazoles, carbonic acid and bicarbonates,
succinic acid, sodium benzoic acid and benzoates, gluconate,
edetate (EDTA), acetate, malate, imidazole, tris, phosphate, and
mixtures thereof. In one embodiment, the buffer is acetate.
[0138] In another embodiment, the buffer is histidine. The
histidine starting material used to prepare the compositions of the
present invention can exist in different forms. For example, the
histidine can be an enantiomeric (e.g., L- or D-enantiomer) or
racemic form of histidine, a free acid or free base form of
histidine, a salt form (e.g., a monohydrochloride, dihydrochloride,
hydrobromide, sulfate, or acetate salt) of histidine, a solvated
form of histidine, a hydrated form (e.g., monohydrate) of
histidine, or an anhydrous form of histidine. The purity of
histidine base and/or salt used to prepare the compositions
generally can be at least about 98%, at least about 99%, or at
least about 99.5%. As used herein, the term "purity" in the context
of histidine refers to chemical purity of histidine as understood
in the art, e.g., as described in The Merck Index, 13th ed., O'Neil
et al. ed. (Merck & Co., 2001).
[0139] When a concentration of a buffer is referred to, it is
intended that the recited concentration represent the molar
concentration of the free acid or free base form of the buffer. For
example, the concentration of the buffer when present in certain
liquid pharmaceutical compositions can range from about 0.1
millimolar (mM) to about 100 mM. In one embodiment, the
concentration of the buffer is from about 1 mM to about 50 mM. In
another embodiment, the concentration of the buffer is from about 5
mM to about 30 mM. In various embodiments, the concentration of the
buffer is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about
20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45
mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70
mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM
or about 100 mM. In one embodiment, the concentration of histidine
in the pharmaceutical composition is about 10 mM. In another
embodiment, the pharmaceutical composition contains about 10 mM of
L-histidine (in base form). In another embodiment, the
concentration of histidine in the pharmaceutical composition is
about 20 mM. In another embodiment, the pharmaceutical composition
contains about 20 mM of L-histidine (in base form). Ranges
intermediate to the above-recited histidine concentrations are also
intended to be part of this invention. For example, ranges of
values using a combination of any of the above-recited values as
upper and/or lower limits are intended to be included.
[0140] In general, the buffer is used to maintain an acceptable pH
level (which can affect antibody stability) in the liquid
pharmaceutical composition. The liquid pharmaceutical composition
typically is buffered to maintain a pH in the range of from about 4
to about 8; from about 4.5 to about 7; from about 5.0 to 6.5, or
from about 5.3 to about 6.3. Ranges intermediate to the
above-recited pH's are also intended to be part of this invention.
For example, ranges of values using a combination of any of the
above-recited values as upper and/or lower limits are intended to
be included. In one embodiment, the liquid pharmaceutical
composition is buffered to maintain a pH of about 5.5. In another
embodiment, the liquid pharmaceutical composition is buffered to
maintain a pH of about 6.0.
[0141] As noted above, the compositions of the present invention
optionally may further comprise a pharmaceutically acceptable
tonicity agent in addition to a chelating agent. As used herein,
the terms "tonicity agent" or "tonicifier" refers to an excipient
that can adjust the osmotic pressure of a liquid antibody
formulation. In certain embodiments, the tonicity agent can adjust
the osmotic pressure of a liquid antibody formulation to isotonic
so that the antibody formulation is physiologically compatible with
the cells of the body tissue of the subject. In still other
embodiments, the "tonicity agent" may contribute to an improvement
in stability of any of the anti-M-CSF antibodies described herein.
An "isotonic" formulation is one that has essentially the same
osmotic pressure as human blood. Isotonic formulations generally
have an osmotic pressure from about 250 to 350 mOsm. The term
"hypotonic" describes a formulation with an osmotic pressure below
that of human blood. Correspondingly, the term "hypertonic" is used
to describe a formulation with an osmotic pressure above that of
human blood. Isotonicity can be measured using a vapor pressure or
ice-freezing type osmometer, for example.
[0142] The tonicity agent used to prepare the compositions of the
present invention can exist in different forms. When the tonicity
agent is referred to, it is intended that all of these different
forms are encompassed by the name of the tonicity agent. For
example, the tonicity agent can be in an enantiomeric (e.g., L- or
D-enantiomer) or racemic form; isomers such as alpha or beta,
including alpha, alpha; or beta, beta; or alpha, beta; or beta,
alpha; a free acid or free base form; a hydrated form (e.g.,
monohydrate), or an anhydrous form.
[0143] In one embodiment, the tonicity agent is a saccharide. As
used herein, the term "saccharide" refers to a class of molecules
that are derivatives of polyhydric alcohols. Saccharides are
commonly referred to as carbohydrates and may contain different
amounts of sugar (saccharide) units, e.g., monosaccharides,
disaccharides and polysaccharides. Saccharides that are suitable
for use as a tonicity agent in the present invention, include, but
are not limited to, saccharides selected from the group consisting
of fructose, glucose, mannose, sorbose, xylose, lactose, maltose,
sucrose, dextran, pullulan, dextrin, cyclodextrins, soluble starch,
hydroxyethyl starch, water-soluble glucans, and mixtures
thereof.
[0144] In another embodiment, the tonicity agent is a polyol. As
used herein, the term "polyol" refers an excipient with multiple
hydroxyl groups, and includes sugars (reducing and nonreducing
sugars), sugar alcohols and sugar acids. In one embodiment, the
polyol has a molecular weight that is less than about 600 kD (e.g.,
in the range from about 120 to about 400 kD). A "reducing sugar" is
one which contains a hemiacetal group that can reduce metal ions or
react covalently with lysine and other amino groups in proteins and
a "nonreducing sugar" is one which does not have these properties
of a reducing sugar. Polyols that are suitable for use as a
tonicity agent in the present invention, include, but are not
limited to, polyols selected from the group consisting of mannitol,
trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol,
xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol,
inositol, and mixtures thereof. In one embodiment, the tonicity
agent is a non-reducing sugar selected from the group consisting of
trehalose, sucrose, and mixtures thereof.
[0145] In one embodiment, the tonicity agent is mannitol. In
another embodiment, the tonicity agent is D-mannitol. In another
embodiment, the tonicity agent is trehalose. In another embodiment,
the tonicity agent is .alpha. .alpha.-trehalose dihydrate. In
another embodiment, the tonicity agent is sucrose.
[0146] In one embodiment, concentration of the tonicity agent in
the liquid pharmaceutical composition ranges from about 1
millimolar to about 600 millimolar, from about 1 millimolar to
about 400 millimolar, from 1 millimolar to about 300 millimolar, or
from 200 millimolar to about 275 millimolar. In one another
embodiment, the tonicity agent is mannitol and is present in the
liquid pharmaceutical composition at a concentration of about 247
millimolar. In another embodiment, the tonicity agent is trehalose
and is present in the liquid pharmaceutical composition at a
concentration of about 222 millimolar. In another embodiment, the
tonicity agent is trehalose and is present in the liquid
pharmaceutical composition at a concentration of about 238
millimolar. In another embodiment, the tonicity agent is sucrose is
present in the liquid pharmaceutical composition at a concentration
of about 263 millimolar.
[0147] In one embodiment, concentration of the tonicity agent in
the liquid pharmaceutical composition ranges from about 1 mg/ml to
about 300 mg/ml, from about 1 mg/ml to about 200 mg/ml, or from
about 50 mg/ml to about 150 mg/ml. In another embodiment, the
tonicity agent is mannitol and is present in the liquid
pharmaceutical composition at a concentration of about 45 mg/ml
millimolar. In another embodiment, the tonicity agent is trehalose
and is present in the liquid pharmaceutical composition at a
concentration of about 84 mg/ml. In another embodiment, the
tonicity agent is trehalose and is present in the liquid
pharmaceutical composition at a concentration of about 90 mg/ml. In
another embodiment, the tonicity agent is sucrose and is present in
the liquid pharmaceutical composition at a concentration of about
90 mg/ml.
[0148] In one embodiment, the tonicity agent is a salt, such as
sodium chloride. In one embodiment, when the tonicity agent is a
salt, the concentration of the salt in the liquid pharmaceutical
composition ranges from about 1 mg/ml to about 20 mg/ml. In another
embodiment, the tonicity agent is sodium chloride and the
concentration of the sodium chloride in the liquid pharmaceutical
composition is about 8.18 mg/ml.
[0149] Ranges intermediate to the above-recited tonicity agent
concentrations are also intended to be part of this invention. For
example, ranges of values using a combination of any of the
above-recited values as upper and/or lower limits are intended to
be included.
[0150] As noted above, the compositions of the present invention
optionally may further comprise a pharmaceutically acceptable
surfactant in addition to a chelating agent. As used herein, the
term "surfactant" refers to an excipient that can alter the surface
tension of a liquid antibody formulation. In certain embodiments,
the surfactant reduces the surface tension of a liquid antibody
formulation. In still other embodiments, the "surfactant" may
contribute to an improvement in stability of any of the anti-M-CSF
antibodies described herein. For example, the surfactant may reduce
aggregation of the formulated antibody and/or minimize the
formation of particulates in the formulation and/or reduces
adsorption. The surfactant may also improve stability of the
antibody during and after a freeze/thaw cycle.
[0151] Suitable surfactants include polysorbate surfactants,
poloxamers (e.g., poloxamer 18 and 407), triton surfactants such as
Triton X-100.RTM., polysorbate surfactants such as Tween 20.RTM.
and Tween 80.RTM., sodium dodecyl sulfate, sodium laurel sulfate,
sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine,
linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine,
myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine,
linoleyl-betaine, myristyl-betaine, cetyl-betaine,
lauroamidopropyl-betaine, cocamidopropyl-betaine,
linoleamidopropyl-betaine, myristamidopropyl-betaine,
palmidopropyl-betaine, isostearamidopropyl-betaine,
myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine,
isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate,
disodium methyl oleyl-taurate, dihydroxypropyl PEG 5 linoleammonium
chloride, polyethylene glycol, polypropylene glycol, and mixtures
thereof.
[0152] In one embodiment, the surfactant is a polysorbate
surfactant comprising at least one excipient that is selected from
the group consisting of polysorbate 20, polysorbate 21, polysorbate
40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80,
polysorbate 81, polysorbate 85, and mixtures thereof. In another
embodiment, the liquid pharmaceutical composition comprises
polysorbate 80.
[0153] The concentration of the surfactant when present in the
liquid pharmaceutical composition generally ranges from about 0.01
mg/ml to about 10 mg/ml, from about 0.05 mg/ml to about 5.0 mg/ml,
from about 0.1 mg/ml to about 1.0 mg/ml, or from about 0.2 mg/ml to
about 0.7 mg/ml. In another embodiment, the concentration of the
surfactant ranges from about 0.05 millimolar to about 1.0
millimolar. In another embodiment, the surfactant is present in an
amount that is about 0.2 mg/ml. In another embodiment, the
surfactant is present in an amount that is about 0.5 mg/ml. In one
embodiment, the liquid pharmaceutical composition contains about
0.2 mg/ml polysorbate 80. In another embodiment, the liquid
pharmaceutical composition contains about 0.4 mg/ml polysorbate 80.
In another embodiment, the liquid pharmaceutical composition
contains about 0.5 mg/ml polysorbate 80.
[0154] Ranges intermediate to the above-recited surfactant
concentrations are also intended to be part of this invention. For
example, ranges of values using a combination of any of the
above-recited values as upper and/or lower limits are intended to
be included.
[0155] As noted above, the compositions of the present invention
optionally may further comprise a pharmaceutically acceptable
antioxidant in addition to a chelating agent. Suitable antioxidants
include, but are not limited to, methionine, sodium thiosulfate,
catalase, and platinum. For example, the liquid pharmaceutical
composition may contain methionine in a concentration that ranges
from 1 mM to about 100 mM, and in particular, is about 27 mM.
[0156] In one embodiment, the present invention encompasses
compositions comprising at least one antibody comprising an amino
acid sequence that is at least 90% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2, and further comprising an
amino acid sequence that is at least 90% identical to a light chain
amino acid sequence shown in SEQ ID NO: 4, wherein the antibody
binds to human M-CSF; and a chelating agent.
[0157] In another embodiment, the present invention encompasses
compositions comprising at least one antibody comprising an amino
acid sequence that is at least 90% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2 without the signal sequence,
and further comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4 without the signal sequence, wherein the antibody binds to human
M-CSF; and a chelating agent.
[0158] In one embodiment, the present invention encompasses
compositions comprising at least one antibody comprising an amino
acid sequence that is at least 95% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2, and further comprising an
amino acid sequence that is at least 95% identical to a light chain
amino acid sequence shown in SEQ ID NO: 4, wherein the antibody
binds to human M-CSF and has a purity of at least about 95%; and a
chelating agent.
[0159] In one embodiment, the present invention encompasses
compositions comprising at least one antibody comprising an amino
acid sequence that is at least 99% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2, and further comprising an
amino acid sequence that is at least 99% identical to a light chain
amino acid sequence shown in SEQ ID NO: 4, wherein the antibody
binds to human M-CSF and has a purity of at least about 95%; and a
chelating agent.
[0160] In one embodiment, the present invention encompasses
compositions comprising at least one antibody comprising an amino
acid sequence that is at least 90% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2, and further comprising an
amino acid sequence that is at least 90% identical to a light chain
amino acid sequence shown in SEQ ID NO: 4, wherein the antibody
binds to human M-CSF and the antibody has a purity of at least
about 90%, 95% or 100%; and a chelating agent.
[0161] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF, and the composition has an antibody concentration of from
about 1.0 mg/ml to about 100 mg/ml.
[0162] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one human
monoclonal IgG2 anti-M-CSF antibody having the heavy and light
chain amino acid sequences of antibody 8.10.3F; and a chelating
agent, wherein the antibody binds to human M-CSF, and the
composition has an antibody concentration of from about 1.0 mg/ml
to about 100 mg/ml.
[0163] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF, and the composition contains a concentration of antibody
that is at least about 5 mg/ml, at least about 10 mg/ml, at least
about 15 mg/ml or at least about 20 mg/ml.
[0164] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF, and the composition contains a concentration of antibody
that is at least about 60 mg/ml, at least about 70 mg/ml, at least
about 80 mg/ml, or at least about 90 mg/ml.
[0165] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF, and the composition contains a concentration of antibody
that is about 10 mg/ml.
[0166] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and a chelating agent, wherein the antibody binds to human
M-CSF, and the composition contains a concentration of antibody
that is about 20 mg/ml.
[0167] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and from about 0.01 millimolar to about 0.5 millimolar of
chelating agent, wherein the antibody binds to human M-CSF, and the
composition has an antibody concentration of from about 1.0 mg/ml
to about 100 mg/ml.
[0168] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and from about 0.01 millimolar to about 0.5 millimolar of
chelating agent, wherein the antibody binds to human M-CSF, and the
composition has an antibody concentration of from about 1.0 mg/ml
to about 100 mg/ml.
[0169] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and about 0.05 millimolar of chelating agent, wherein the
antibody binds to human M-CSF, and the composition has an antibody
concentration of from about 1.0 mg/ml to about 100 mg/ml.
[0170] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and from about 0.01 millimolar to about 0.5 millimolar of EDTA,
wherein the antibody binds to human M-CSF, and the composition has
an antibody concentration of from about 1.0 mg/ml to about 100
mg/ml.
[0171] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; and from about 1.0 millimolar to about 100 millimolar of
histidine, wherein the antibody binds to human M-CSF, and the
composition has an antibody concentration of from about 1.0 mg/ml
to about 100 mg/ml.
[0172] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; from about 0.01 millimolar to about 0.5 millimolar of EDTA; and
from about 1 millimolar to about 50 millimolar of histidine,
wherein the antibody binds to human M-CSF, and the composition has
an antibody concentration of from about 1.0 mg/ml to about 100
mg/ml.
[0173] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; from about 0.01 millimolar to about 0.5 millimolar of EDTA; from
about 1 millimolar to about 50 millimolar of histidine; and from
about 200 millimolar to about 300 millimolar of mannitol, wherein
the antibody binds to human M-CSF, and the composition has an
antibody concentration of from about 1.0 mg/ml to about 100
mg/ml.
[0174] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; from about 0.01 millimolar to about 0.5 millimolar of EDTA; from
about 0.1 millimolar to about 50 millimolar of histidine; and from
about 200 millimolar to about 300 millimolar of trehalose, wherein
the antibody binds to human M-CSF, and the composition has an
antibody concentration of from about 1.0 mg/ml to about 100
mg/ml.
[0175] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 50 millimolar of a chelating agent.
[0176] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 5.0 millimolar of a chelating agent.
[0177] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and about 0.05 millimolar of a
chelating agent.
[0178] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 50 millimolar of EDTA.
[0179] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 10.0 millimolar of EDTA.
[0180] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 0.01 millimolar to
about 1.0 millimolar of EDTA.
[0181] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and about 0.05 millimolar of
EDTA.
[0182] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 30 micromolar to
about 5.0 millimolar of DTPA.
[0183] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 5.0 millimolar of deferoxamine.
[0184] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 mM to about 100 mM
of histidine.
[0185] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 30 micromolar to about
5.0 millimolar of a chelating agent; and from about 1 mM to about
100 mM of histidine.
[0186] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; and from about 10 millimolar
to about 400 millimolar of trehalose.
[0187] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; and from about 1 millimolar to
about 400 millimolar of mannitol.
[0188] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; from about 10 millimolar to
about 400 millimolar of trehalose; and from about 1 mM to about 100
mM of histidine.
[0189] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; from about 10 millimolar to
about 400 millimolar of trehalose; from about 1 mM to about 100 mM
of histidine; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0190] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; from about 1 millimolar to
about 400 millimolar of mannitol; from about 1 mM to about 100 mM
of histidine; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0191] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of a tonicity agent; from about 1 mM to about 100 mM of
a buffer; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0192] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of a tonicity agent; from about 1 mM to about 100 mM of
histidine; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0193] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of a monoclonal
anti-M-CSF antibody; from about 1 micromolar to about 5.0
millimolar of EDTA; from about 10 millimolar to about 400
millimolar of trehalose; from about 1 mM to about 100 mM of
histidine; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0194] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of mannitol; from about 1 mM to about 100 mM of
histidine; and from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80.
[0195] In certain aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 0.1 mg/ml to
about 200 mg/ml of a monoclonal anti-M-CSF antibody; from about 1
mM to about 100 mM of histidine; from about 0.005 millimolar to
about 10 millimolar of polysorbate 80; from about 1 micromolar to
about 5.0 millimolar of EDTA; and from about 10 millimolar to about
400 millimolar of trehalose.
[0196] In certain aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 0.1 mg/ml to
about 200 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 1 mM to about 100 mM of histidine; from about 0.01 mg/ml
to about 10 mg/ml of polysorbate 80; from about 1 micromolar to
about 5.0 millimolar of EDTA; and from about 10 millimolar to about
400 millimolar of mannitol.
[0197] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 1.0 mg/ml to
about 100 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 10 mM to about 50 mM of histidine; from about 0.05 mg/ml
to about 1 mg/ml of polysorbate 80; from about 0.01 millimolar to
about 1 millimolar of EDTA; and from about 100 millimolar to about
300 millimolar of trehalose.
[0198] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 1.0 mg/ml to
about 100 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 10 mM to about 50 mM of histidine; from about 0.05 mg/ml
to about 1 mg/ml of polysorbate 80; from about 0.01 millimolar to
about 1 millimolar of EDTA; and from about 100 millimolar to about
300 millimolar of mannitol.
[0199] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 10 mg/ml to
about 50 mg/ml of at least one monoclonal anti-M-CSF antibody; from
about 10 mM to about 30 mM of histidine; from about 0.05 millimolar
to about 0.2 mg/ml; from about 0.01 millimolar to about 1
millimolar of EDTA; and from about 200 millimolar to about 250
millimolar of trehalose.
[0200] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise about 20 mg/ml of at
least one monoclonal anti-M-CSF antibody; about 20 mM of histidine;
about 0.15 millimolar of polysorbate 80; about 0.05 millimolar of
EDTA; and about 222 millimolar of trehalose.
[0201] In other aspects of the present invention, the composition
comprises at least one monoclonal anti-M-CSF antibody; histidine;
polysorbate 80; EDTA; and mannitol.
[0202] In still other aspects of the present invention, the
composition comprises monoclonal anti-M-CSF antibody; histidine;
polysorbate 80; EDTA; and trehalose.
[0203] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising at least one anti-M-CSF
antibody and DTPA.
[0204] In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, a
chelating agent, and a buffer. In another embodiment, the invention
is directed to a composition comprising at least one anti-M-CSF
antibody, a chelating agent, and histidine. In another embodiment,
the invention is directed to a composition comprising at least one
anti-M-CSF antibody, EDTA, and histidine. In another embodiment,
the invention is directed to a composition comprising at least one
anti-M-CSF antibody, DTPA, and histidine.
[0205] In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, a
chelating agent, and a buffer. In another embodiment, the invention
is directed to a composition comprising at least one anti-M-CSF
antibody, a chelating agent, and acetate.
[0206] In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, a
chelating agent, and a tonicity agent. In another embodiment, the
invention is directed to a liquid pharmaceutical composition
comprising at least one anti-M-CSF antibody, a chelating agent, and
mannitol. In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, a
chelating agent, and trehalose. In another embodiment, the
invention is directed to a composition comprising at least one
anti-M-CSF antibody, EDTA, and trehalose. In another embodiment,
the invention is directed to a composition comprising at least one
anti-M-CSF antibody, EDTA, and mannitol. In another embodiment, the
invention is directed to a composition comprising at least one
anti-M-CSF antibody, EDTA, and sucrose. In another embodiment, the
invention is directed to a composition comprising at least one
anti-M-CSF antibody, DTPA, and trehalose. In another embodiment,
the invention is directed to a composition comprising at least one
anti-M-CSF antibody, DTPA, and mannitol. In another embodiment, the
invention is directed to a composition comprising at least one
anti-M-CSF antibody, a chelating agent, and a surfactant. In
another embodiment, the invention is directed to a composition
comprising at least one anti-M-CSF antibody, EDTA, and a
surfactant. In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, DTPA, and
a surfactant. In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody, a
chelating agent selected from the group consisting of EDTA and
DTPA, and polysorbate 80. In another embodiment, the invention is
directed to a composition comprising at least one anti-M-CSF
antibody, a buffer, and a surfactant. In another embodiment, the
invention is directed to a composition comprising at least one
anti-M-CSF antibody, histidine, and a surfactant. In another
embodiment, the invention is directed to a composition comprising
at least one anti-M-CSF antibody, histidine, and polysorbate 80. In
another embodiment, the invention is directed to a composition
comprising at least one anti-M-CSF antibody, a chelating agent, a
buffer, and a surfactant. In another embodiment, the invention is
directed to a composition comprising at least one anti-M-CSF
antibody, a chelating agent, a buffer, and a tonicity agent. In
another embodiment, the invention is directed to a composition
comprising at least one anti-M-CSF antibody, a chelating agent, a
buffer, a surfactant, and a tonicity agent. In another embodiment,
the invention is directed to a composition comprising at least one
anti-M-CSF antibody and histidine. In another embodiment, the
invention is directed to a liquid pharmaceutical composition
comprising at least one anti-M-CSF antibody and histidine.
[0207] In certain embodiments, the anti-M-CSF antibody compositions
comprise at least one anti-M-CSF antibody, histidine, and EDTA. In
other embodiments, the anti-M-CSF antibody compositions comprise at
least one anti-M-CSF antibody, histidine, and polysorbate 80. In
other embodiments, the anti-M-CSF antibody compositions comprise at
least one anti-M-CSF antibody, histidine, and mannitol. In other
embodiments, the anti-M-CSF antibody compositions comprise at least
one anti-M-CSF antibody, histidine, and trehalose. In other
embodiments, the anti-M-CSF antibody compositions comprise at least
one anti-M-CSF antibody, histidine, and sucrose. In other
embodiments, the anti-M-CSF antibody compositions comprise at least
one anti-M-CSF antibody, histidine, polysorbate 80, and EDTA. In
other embodiments, the anti-M-CSF antibody compositions comprise at
least one anti-M-CSF antibody, histidine, polysorbate 80, mannitol,
and EDTA. In other embodiments, the anti-M-CSF antibody
compositions comprise at least one anti-M-CSF antibody, histidine,
polysorbate 80, trehalose, and EDTA. In other embodiments, the
anti-M-CSF antibody compositions comprise at least one anti-M-CSF
antibody, histidine, polysorbate 80, sucrose, and EDTA.
[0208] In still further embodiments, the anti-M-CSF antibody
compositions comprise at least one anti-M-CSF antibody, acetate,
and EDTA. In other embodiments, the M-CSF antibody compositions
comprise at least one anti-M-CSF antibody, polysorbate 80, and
EDTA. In other embodiments, the anti-M-CSF antibody compositions
comprise at least one anti-M-CSF antibody, mannitol, and EDTA. In
other embodiments, the anti-M-CSF antibody compositions comprise at
least one anti-M-CSF antibody, polysorbate 80, mannitol, and EDTA.
In other embodiments, the anti-M-CSF antibody compositions comprise
at least one anti-M-CSF antibody, acetate, polysorbate 80,
mannitol, and EDTA. In other embodiments, the anti-M-CSF antibody
compositions comprise at least one anti-M-CSF antibody, sucrose,
and EDTA. In other embodiments, the anti-M-CSF antibody
compositions comprise at least one anti-M-CSF antibody, polysorbate
80, sucrose, and EDTA. In other embodiments, the anti-M-CSF
antibody compositions comprise at least one anti-M-CSF antibody,
acetate, polysorbate 80, sucrose, and EDTA. In other embodiments,
the anti-M-CSF antibody compositions comprise at least one
anti-M-CSF antibody, trehalose, and EDTA. In other embodiments, the
anti-M-CSF antibody compositions comprise at least one anti-M-CSF
antibody, polysorbate 80, trehalose, and EDTA. In other
embodiments, the anti-M-CSF antibody compositions comprise at least
one anti-M-CSF antibody, acetate, polysorbate 80, trehalose, and
EDTA.
[0209] In certain aspects of the present invention, the anti-M-CSF
antibody compositions comprise from about 1 mg/ml to about 100
mg/ml of at least one monoclonal anti-M-CSF antibody; from about 1
mM to about 50 mM of histidine; from about 0.01 mg/ml to about 5
mg/ml of polysorbate 80; from about 0.001 mg/ml to about 0.5 mg/ml
of EDTA; and from about 10 mg/ml to about 200 mg/ml of
mannitol.
[0210] In certain aspects of the present invention, the anti-M-CSF
antibody compositions comprise from about 1 mg/ml to about 100
mg/ml of at least one monoclonal anti-M-CSF antibody; from about 1
mM to about 50 mM of histidine; from about 0.01 mg/ml to about 5
mg/ml of polysorbate 80; from about 0.001 mg/ml to about 0.5 mg/ml
of EDTA; and from about 10 mg/ml to about 200 mg/ml of
trehalose.
[0211] In certain aspects of the present invention, the anti-M-CSF
antibody compositions comprise from about 1 mg/ml to about 100
mg/ml of at least one monoclonal anti-M-CSF antibody; from about 1
mM to about 50 mM of histidine; from about 0.01 mg/ml to about 5
mg/ml of polysorbate 80; from about 0.001 mg/ml to about 0.5 mg/ml
of EDTA; and from about 10 mg/ml to about 200 mg/ml of sucrose.
[0212] In other aspects of the present invention, the anti-M-CSF
antibody compositions comprise about 10 mg/ml of antibody; about 10
mM of histidine; about 0.2 mg/ml of polysorbate 80; about 0.02
mg/ml of EDTA; and about 45 mg/ml of mannitol.
[0213] In other aspects of the present invention, the anti-M-CSF
antibody compositions comprise about 75 mg/ml of antibody; about 20
mM of histidine; about 0.5 mg/ml of polysorbate 80; about 0.05
mg/ml of EDTA; and about 90 mg/ml of sucrose.
[0214] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 0.3 micromolar to
about 50 millimolar of a chelating agent.
[0215] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 1 millimolar of a chelating agent.
[0216] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and about 0.02 millimolar of a
chelating agent.
[0217] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and about 0.05 millimolar of a
chelating agent.
[0218] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 50 millimolar of EDTA.
[0219] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-M-CSF antibody; and from about 1 micromolar to about 10.0
millimolar of EDTA.
[0220] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 0.01 millimolar to
about 1.0 millimolar of EDTA.
[0221] In another embodiment, the composition comprises from about
0.1 mg/ml to about 100 mg/ml of at least one monoclonal anti-M-CSF
antibody; and about 0.02 millimolar of EDTA.
[0222] In another embodiment, the composition comprises from about
0.1 mg/ml to about 100 mg/ml of at least one monoclonal anti-M-CSF
antibody; and about 0.05 millimolar of EDTA.
[0223] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 30 micromolar to
about 5.0 millimolar of DTPA.
[0224] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 micromolar to
about 5.0 millimolar of deferoxamine.
[0225] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; and from about 1 mM to about 100 mM
of histidine.
[0226] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
5.0 millimolar of a chelating agent; and from about 1 mM to about
100 mM of histidine.
[0227] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of a chelating agent; and from about 10 millimolar
to about 400 millimolar of trehalose.
[0228] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of a chelating agent; and from about 1 millimolar to
about 400 millimolar of mannitol.
[0229] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of a chelating agent; from about 10 millimolar to
about 400 millimolar of trehalose; and from about 1 mM to about 100
mM of histidine.
[0230] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of a chelating agent; from about 10 millimolar to
about 400 millimolar of trehalose; from about 1 mM to about 100 mM
of histidine; and from about 0.01 mg/ml to about 10 mg/ml of a
surfactant.
[0231] In another embodiment, the composition comprises from about
0.1 mg/ml to about 200 mg/ml of at least one monoclonal anti-M-CSF
antibody; from about 1 micromolar to about 1.0 millimolar of a
chelating agent; from about 1 millimolar to about 400 millimolar of
mannitol; from about 1 mM to about 100 mM of histidine; and from
about 0.01 mg/ml to about 10 mg/ml of a surfactant.
[0232] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of a tonicity agent; from about 1 mM to about 100 mM of
a buffer; and from about 0.01 mg/ml to about 10 mg/ml of a
surfactant.
[0233] In another embodiment, the composition comprises from about
0.1 mg/ml to about 200 mg/ml of at least one monoclonal anti-M-CSF
antibody; from about 1 micromolar to about 1.0 millimolar of EDTA;
from about 10 millimolar to about 400 millimolar of a tonicity
agent; from about 1 mM to about 100 mM of histidine; and from about
0.01 mg/ml to about 10 mg/ml of a surfactant.
[0234] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of trehalose; from about 1 mM to about 100 mM of
histidine; and from about 0.1 mg/ml to about 1 mg/ml of a
surfactant.
[0235] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of at least one
monoclonal anti-M-CSF antibody; from about 1 micromolar to about
1.0 millimolar of EDTA; from about 10 millimolar to about 400
millimolar of mannitol; from about 1 mM to about 100 mM of
histidine; and from about from about 0.1 mg/ml to about 1 mg/ml of
a surfactant.
[0236] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 0.1 mg/ml to
about 200 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 1 mM to about 100 mM of histidine; from about 0.005
millimolar to about 10 millimolar of polysorbate 80; from about 1
micromolar to about 1.0 millimolar of EDTA; and from about 10
millimolar to about 400 millimolar of trehalose.
[0237] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 0.1 mg/ml to
about 200 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 1 mM to about 100 mM of histidine; from about 0.005
millimolar to about 10 millimolar of polysorbate 80; from about 1
micromolar to about 1.0 millimolar of EDTA; and from about 10
millimolar to about 400 millimolar of mannitol.
[0238] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 1.0 mg/ml to
about 100 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 10 mM to about 50 mM of histidine; from about 0.01
millimolar to about 1.0 millimolar of polysorbate 80; from about 1
micromolar to about 1.0 millimolar of EDTA; and from about 100
millimolar to about 300 millimolar of trehalose.
[0239] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 1.0 mg/ml to
about 100 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 10 mM to about 50 mM of histidine; from about 0.01
millimolar to about 1.0 millimolar of polysorbate 80; from about 1
micromolar to about 1.0 millimolar of EDTA; and from about 100
millimolar to about 300 millimolar of mannitol.
[0240] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 10 mg/ml to
about 50 mg/ml of at least one monoclonal anti-M-CSF antibody; from
about 10 mM to about 30 mM of histidine; from about 0.05 millimolar
to about 0.5 millimolar of polysorbate 80; from about 0.1
millimolar to about 1 millimolar of EDTA; and from about 200
millimolar to about 250 millimolar of trehalose.
[0241] In other aspects of the present invention, the liquid
anti-M-CSF antibody compositions comprise from about 50 mg/ml to
about 100 mg/ml of at least one monoclonal anti-M-CSF antibody;
from about 10 mM to about 30 mM of histidine; from about 0.05
millimolar to about 1 millimolar of polysorbate 80; from about 0.01
millimolar to about 1 millimolar of EDTA; and from about 100
millimolar to about 400 millimolar of sucrose.
[0242] In other aspects of the present invention, the anti-M-CSF
liquid antibody compositions comprise about 10 mg/ml of at least
one monoclonal anti-M-CSF antibody; about 10 mM of histidine; about
0.2 mg/ml of polysorbate 80; about 0.02 mg/ml of EDTA; and about 45
mg/ml of mannitol.
[0243] In other aspects of the present invention, the anti-M-CSF
antibody compositions comprise about 75 mg/ml of at least one
monoclonal anti-M-CSF antibody; about 20 mM of histidine; about 0.5
mg/ml of polysorbate 80; about 0.05 millimolar of EDTA; and about
90 mg/ml of sucrose.
[0244] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising at least one anti-M-CSF
antibody and a chelating agent, wherein the molar concentration of
the antibody ranges from about 0.01 millimolar to about 2
millimolar and the molar concentration of the chelating agent
ranges from about 0.001 millimolar to about 5 millimolar, and
wherein the molar ratio of antibody to chelating agent ranges from
about 0.002 to about 2000; from about 0.01 to about 500; from about
0.05 to about 100; from about 0.1 to about 50; from about 0.5 to
about 10; from about 1 to about 5; from about 1 to about 3; or
about 1.6.
[0245] In another embodiment, the invention is directed to a
composition comprising at least one anti-M-CSF antibody and a
chelating agent, wherein the molar concentration of the antibody
ranges from about 0.01 millimolar to about 2 millimolar and the
molar concentration of the chelating agent ranges from about 0.001
millimolar to about 5 millimolar, and wherein the molar ratio of
antibody to chelating agent ranges from about 0.002 to about 2000;
from about 0.01 to about 500; from about 0.05 to about 100; from
about 0.1 to about 50; from about 0.5 to about 10; from about 1 to
about 5; or about 3.8.
[0246] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising at least one
monoclonal anti-M-CSF antibody 8.10.3F and a chelating agent,
wherein the molar concentration of the antibody ranges from about
0.01 millimolar to about 2 millimolar and the molar concentration
of the chelating agent ranges from about 0.001 millimolar to about
5 millimolar, and wherein the molar ratio of antibody to chelating
agent ranges from about 0.002 to about 2000; from about 0.01 to
about 500; from about 0.05 to about 100; from about 0.1 to about
50; from about 0.5 to about 10; from about 1 to about 5; from about
1 to about 3; or about 1.6.
[0247] In another embodiment, the invention is directed to a stable
composition comprising at least one monoclonal anti-M-CSF antibody
8.10.3F and a chelating agent, wherein the molar concentration of
the antibody ranges from about 0.01 millimolar to about 2
millimolar and the molar concentration of the chelating agent
ranges from about 0.001 millimolar to about 5 millimolar, and
wherein the molar ratio of antibody to chelating agent ranges from
about 0.002 to about 2000; from about 0.01 to about 500; from about
0.05 to about 100; from about 0.1 to about 50; from about 0.5 to
about 10; from about 1 to about 5; or about 3.8.
[0248] In another embodiment, the invention is directed to a stable
composition comprising at least one monoclonal anti-M-CSF antibody
8.10.3F, a chelating agent, and histidine; wherein the molar
concentration of the antibody ranges from about 0.01 millimolar to
about 2 millimolar and the molar concentration of histidine ranges
from about 1 millimolar to about 100 millimolar and the molar
concentration of the chelating agent ranges from about 0.001
millimolar to about 5 millimolar, and wherein the molar ratio of
antibody to chelating agent ranges from about 0.002 to about 2000;
from about 0.01 to about 500; from about 0.05 to about 100; from
about 0.1 to about 50; from about 0.5 to about 10; from about 1 to
about 5; or about 3.8.
[0249] In another embodiment, the invention is directed to a stable
composition comprising at least one monoclonal anti-M-CSF antibody
8.10.3F, a chelating agent, and histidine; wherein the molar
concentration of the antibody ranges from about 0.01 millimolar to
about 2 millimolar and the molar concentration of histidine ranges
from about 5 millimolar to about 30 millimolar and the molar
concentration of the chelating agent ranges from about 0.001
millimolar to about 5 millimolar, and wherein the molar ratio of
antibody to chelating agent ranges from about 0.002 to about 2000;
from about 0.01 to about 500; from about 0.05 to about 100; from
about 0.1 to about 50; from about 0.5 to about 10; from about 1 to
about 5; or about 3.8.
[0250] In another embodiment, the invention is directed to a stable
composition comprising at least one monoclonal anti-M-CSF antibody
8.10.3F, a chelating agent, and histidine; wherein the molar
concentration of the antibody ranges from about 0.01 millimolar to
about 2 millimolar and the molar concentration of histidine is
about 20 millimolar and the molar concentration of the chelating
agent ranges from about 0.001 millimolar to about 5 millimolar, and
wherein the molar ratio of antibody to chelating agent ranges from
about 0.002 to about 2000; from about 0.01 to about 500; from about
0.05 to about 100; from about 0.1 to about 50; from about 0.5 to
about 10; from about 1 to about 5; or about 3.8.
Methods of Producing Anti-M-CSF Antibodies and Antibody Producing
Cell Lines:
[0251] Antibodies in accordance with the invention can be prepared
through the utilization of a transgenic mouse that has a
substantial portion of the human antibody producing genome
inserted, but that is rendered deficient in the production of
endogenous, murine, antibodies. Such mice, then, are capable of
producing human immunoglobulin molecules and antibodies and are
deficient in the production of murine immunoglobulin molecules and
antibodies. Technologies utilized for achieving the same are
discussed below.
[0252] It is possible to produce transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. In particular, however, one embodiment of transgenic
production of mice and antibodies therefrom is disclosed in U.S.
Published Application No. 20050059113 to Bedian, et al. Through use
of such technology, antibodies that bind to M-CSF and hybridomas
producing such antibodies can be prepared.
[0253] Human antibodies avoid potential problems associated with
antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a subject
that receives administration of such antibodies.
[0254] For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
(e.g., M-CSF) challenge. See, e.g., Jakobovits et al, Proc. Natl.
Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,
362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33
(1993); and Duchosal et al., Nature 355:258 (1992). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech
14:309 (1996)).
[0255] In some embodiments, human antibodies are produced by
immunizing a non-human animal comprising in its genome some or all
of human immunoglobulin heavy chain and light chain loci with an
M-CSF antigen. In a preferred embodiment, the non-human animal is a
XENOMOUSE.TM. animal (Abgenix Inc., Fremont, Calif.). Another
non-human animal that may be used is a transgenic mouse produced by
Medarex (Medarex, Inc., Princeton, N.J.).
[0256] In some embodiments, human anti-M-CSF antibodies can be
produced by immunizing a non-human transgenic animal, e.g.,
XENOMOUSE.TM. mice, whose genome comprises human immunoglobulin
genes so that the recombinant mouse produces human antibodies.
XENOMOUSE.TM. mice are engineered mouse strains that comprise large
fragments of human immunoglobulin heavy chain and light chain loci
and are deficient in mouse antibody production. XENOMOUSE.TM. mice
produce an adult-like human repertoire of fully human antibodies
and generate antigen-specific human antibodies. In some
embodiments, the XENOMOUSE.TM. mice contain approximately 80% of
the human antibody V gene repertoire through introduction of
megabase sized, germline configuration yeast artificial chromosome
(YAC) fragments of the human heavy chain loci and kappa light chain
loci. In other embodiments, XENOMOUSE.TM. mice further contain
approximately all of the lambda light chain locus. See, e.g., Green
et al., Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos.
5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001,
6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO
91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO
98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, and
WO 00/037504.
[0257] In some embodiments, the non-human animal comprising human
immunoglobulin genes are animals that have a human immunoglobulin
"minilocus". In the minilocus approach, an exogenous Ig locus is
mimicked through the inclusion of individual genes from the Ig
locus. Thus, one or more V.sub.H genes, one or more D.sub.H genes,
one or more J.sub.H genes, a mu constant domain, and a second
constant domain (preferably a gamma constant domain) are formed
into a construct for insertion into an animal. This approach is
described, inter alia, in U.S. Pat. Nos. 5,545,807, 5,545,806,
5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650,
5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and
5,643,763.
[0258] Therefore, in some embodiments, human antibodies can be
produced by immunizing a non-human animal comprising in its genome
some or all of human immunoglobulin heavy chain and light chain
loci with an M-CSF antigen.
[0259] In some embodiments, the M-CSF antigen is isolated and/or
purified M-CSF. In a preferred embodiment, the M-CSF antigen is
human M-CSF. In some embodiments, the M-CSF antigen is a fragment
of M-CSF. In some embodiments, the M-CSF fragment comprises at
least one epitope of M-CSF. In other embodiments, the M-CSF antigen
is a cell that expresses or overexpresses M-CSF or an immunogenic
fragment thereof on its surface. In still other embodiments, the
M-CSF antigen is an M-CSF fusion protein. M-CSF can be purified
from natural sources using known techniques. In addition,
recombinant M-CSF protein is commercially available.
[0260] In a preferred embodiment, the non-human animal is a
XENOMOUSE.TM. animal (Abgenix Inc., Fremont, Calif.). Another
non-human animal that may be used is a transgenic mouse produced by
Medarex (Medarex, Inc., Princeton, N.J.).
[0261] Immunization of animals can be by any method known in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Press, 1990. Methods for immunizing
non-human animals such as mice, rats, sheep, goats, pigs, cattle
and horses are well known in the art. See, e.g., Harlow and Lane,
supra, and U.S. Pat. No. 5,994,619. In a preferred embodiment, the
M-CSF antigen is administered with an adjuvant to stimulate the
immune response. Exemplary adjuvants include complete or incomplete
Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
(immunostimulating complexes). Such adjuvants may protect the
polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Preferably, if a polypeptide is
being administered, the immunization schedule can involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0262] After immunization of an animal with an M-CSF antigen,
antibodies and/or antibody-producing cells can be obtained from the
animal. In some embodiments, anti-M-CSF antibody-containing serum
is obtained from the animal by bleeding or sacrificing the animal.
The serum may be used as it is obtained from the animal, an
immunoglobulin fraction may be obtained from the serum, or the
anti-M-CSF antibodies may be purified from the serum.
[0263] In some embodiments, antibody-producing immortalized cell
lines are prepared from cells isolated from the immunized animal.
After immunization, the animal is sacrificed and lymph node and/or
splenic B cells are immortalized. Methods of immortalizing cells
include, but are not limited to, transfecting them with oncogenes,
infecting them with an oncogenic virus, cultivating them under
conditions that select for immortalized cells, subjecting them to
carcinogenic or mutating compounds, fusing them with an
immortalized cell, e.g., a myeloma cell, and inactivating a tumor
suppressor gene. See, e.g., Harlow and Lane, supra. In a preferred
embodiment, the immunized animal is a non-human animal that
expresses human immunoglobulin genes and the splenic B cells are
fused to a myeloma cell line from the same species as the non-human
animal. In a more preferred embodiment, the immunized animal is a
XENOMOUSE.TM. animal and the myeloma cell line is a non-secretory
mouse myeloma. In an even more preferred embodiment, the myeloma
cell line is P3-X63-AG8-653. If fusion with myeloma cells is used,
the myeloma cells preferably do not secrete immunoglobulin
polypeptides (a non-secretory cell line). Immortalized cells are
screened using M-CSF, a portion thereof, or a cell expressing
M-CSF. In a preferred embodiment, the initial screening is
performed using an enzyme-linked immunoassay (ELISA) or a
radioimmunoassay. An example of ELISA screening is provided in WO
00/37504.
[0264] Anti-M-CSF antibody-producing cells, e.g., hybridomas, are
selected, cloned and further screened for desirable
characteristics, including robust growth, high antibody production
and desirable antibody characteristics, as discussed further below.
Hybridomas can be expanded in vivo in syngeneic animals, in animals
that lack an immune system, e.g., nude mice, or in cell culture in
vitro. Methods of selecting, cloning and expanding hybridomas are
well known to those of ordinary skill in the art.
[0265] As will be appreciated, antibodies in accordance with the
present invention can be recombinantly expressed in cell lines
other than hybridoma cell lines. Nucleic acid sequences encoding
the cDNAs or genomic clones for the particular antibodies can be
used for transformation of a suitable mammalian or nonmammalian
host cells.
[0266] The present invention also encompasses nucleic acid
molecules encoding anti-M-CSF antibodies. In some embodiments,
different nucleic acid molecules encode a heavy chain and a light
chain of an anti-M-CSF immunoglobulin. In other embodiments, the
same nucleic acid molecule encodes a heavy chain and a light chain
of an anti-M-CSF immunoglobulin. In one embodiment, the nucleic
acid encodes an anti-M-CSF antibody of the invention.
[0267] A nucleic acid molecule encoding the heavy or entire light
chain of an anti-M-CSF antibody or portions thereof can be isolated
from any source that produces such antibody. In various
embodiments, the nucleic acid molecules are isolated from a B cell
isolated from an animal immunized with anti-M-CSF or from an
immortalized cell derived from such a B cell that expresses an
anti-M-CSF antibody. Methods of isolating mRNA encoding an antibody
are well-known in the art. See, e.g., Sambrook, et al., Molecular
Cloning 3rd Ed. Vol. 3 (1989). The mRNA may be used to produce cDNA
for use in the polymerase chain reaction (PCR) or cDNA cloning of
antibody genes. In a preferred embodiment, the nucleic acid
molecule is isolated from a hybridoma that has as one of its fusion
partners a human immunoglobulin-producing cell from a non-human
transgenic animal. In an even more preferred embodiment, the human
immunoglobulin producing cell is isolated from a XENOMOUSE.TM.
animal. In another embodiment, the human immunoglobulin-producing
cell is from a non-human, non-mouse transgenic animal, as described
above. In another embodiment, the nucleic acid is isolated from a
non-human, non-transgenic animal. The nucleic acid molecules
isolated from a non-human animal may be used, e.g., for humanized
antibodies.
[0268] In some embodiments, a nucleic acid encoding a heavy chain
of an anti-M-CSF antibody of the invention can comprise a
polynucleotide sequence encoding a V.sub.H domain of the invention
joined in-frame to a polynucleotide sequence encoding a heavy chain
constant domain from any source. Similarly, a nucleic acid molecule
encoding a light chain of an anti-M-CSF antibody of the invention
can comprise a polynucleotide sequence encoding a V.sub.L domain of
the invention joined in-frame to a polynucleotide sequence encoding
a light chain constant domain from any source.
[0269] In a further aspect of the invention, nucleic acid molecules
encoding the variable domain of the heavy (V.sub.H) and light
(V.sub.L) chains are "converted" to full-length antibody genes. In
one embodiment, nucleic acid molecules encoding the V.sub.H or
V.sub.L domains are converted to full-length antibody genes by
insertion into an expression vector already encoding heavy chain
constant (C.sub.H) or light chain (C.sub.L) constant domains,
respectively, such that the V.sub.H segment is operatively linked
to the C.sub.H segment(s) within the vector, and the V.sub.L
segment is operatively linked to the C.sub.L segment within the
vector. In another embodiment, nucleic acid molecules encoding the
V.sub.H and/or V.sub.L domains are converted into full-length
antibody genes by linking, e.g., ligating, a nucleic acid molecule
encoding a V.sub.H and/or V.sub.L domains to a nucleic acid
molecule encoding a C.sub.H and/or C.sub.L domain using standard
molecular biological techniques. Nucleic acid sequences of human
heavy and light chain immunoglobulin constant domain genes are
known in the art. See, e.g., Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991.
Nucleic acid molecules encoding the full-length heavy and/or light
chains may then be expressed from a cell into which they have been
introduced and the anti-M-CSF antibody isolated.
[0270] The present invention also provides vectors comprising
nucleic acid molecules that encode the heavy chain of an anti-M-CSF
antibody of the invention or an antigen-binding portion thereof.
The invention also provides vectors comprising nucleic acid
molecules that encode the light chain of such antibodies or
antigen-binding portion thereof. The invention further provides
vectors comprising nucleic acid molecules encoding fusion proteins,
modified antibodies, antibody fragments, and probes thereof.
[0271] In some embodiments, the anti-M-CSF antibodies, or
antigen-binding portions of the invention are expressed by
inserting DNAs encoding partial or full-length light and heavy
chains, obtained as described above, into expression vectors such
that the genes are operatively linked to necessary expression
control sequences such as transcriptional and translational control
sequences. Expression vectors include plasmids, retroviruses,
adenoviruses, adeno-associated viruses (AAV), plant viruses such as
cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV
derived episomes, and the like. The antibody gene is ligated into a
vector such that transcriptional and translational control
sequences within the vector serve their intended function of
regulating the transcription and translation of the antibody gene.
The expression vector and expression control sequences are chosen
to be compatible with the expression host cell used. The antibody
light chain gene and the antibody heavy chain gene can be inserted
into separate vectors. In a preferred embodiment, both genes are
inserted into the same expression vector. The antibody genes are
inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites
are present).
[0272] A convenient vector is one that encodes a functionally
complete human C.sub.H or C.sub.L immunoglobulin sequence, with
appropriate restriction sites engineered so that any V.sub.H or
V.sub.L sequence can easily be inserted and expressed, as described
above. In such vectors, splicing usually occurs between the splice
donor site in the inserted J region and the splice acceptor site
preceding the human C domain, and also at the splice regions that
occur within the human C.sub.H exons. Polyadenylation and
transcription termination occur at native chromosomal sites
downstream of the coding regions. The recombinant expression vector
also can encode a signal peptide that facilitates secretion of the
antibody chain from a host cell. The antibody chain gene may be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the immunoglobulin chain. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0273] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
It will be appreciated by those skilled in the art that the design
of the expression vector, including the selection of regulatory
sequences may depend on such factors as the choice of the host cell
to be transformed, the level of expression of protein desired, etc.
Preferred regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from retroviruses (such as retroviral LTRs),
cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian
Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus,
(e.g., the adenovirus major late promoter (AdMLP)), polyoma and
strong mammalian promoters such as native immunoglobulin and actin
promoters. For further description of viral regulatory elements,
and sequences thereof, see e.g., U.S. Pat. No. 5,168,062, U.S. Pat.
No. 4,510,245 and U.S. Pat. No. 4,968,615. Methods for expressing
antibodies in plants, including a description of promoters and
vectors, as well as transformation of plants is known in the art.
See, e.g., U.S. Pat. No. 6,517,529, herein incorporated by
reference. Methods of expressing polypeptides in bacterial cells or
fungal cells, e.g., yeast cells, are also well known in the
art.
[0274] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells
with methotrexate selection/amplification), the neomycin resistance
gene (for G418 selection), and the glutamine synthetase gene.
[0275] Nucleic acid molecules encoding anti-M-CSF antibodies and
vectors comprising these nucleic acid molecules can be used for
transformation of a suitable mammalian, plant, bacterial or yeast
host cell. Antibodies of the invention can be produced
transgenically through the generation of a mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form
therefrom.
[0276] Transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example packaging
the polynucleotide in a virus (or into a viral vector) and
transducing a host cell with the virus (or vector) or by
transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The
transformation procedure used depends upon the host to be
transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include, but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, particle bombardment,
encapsulation of the polynucleotide(s) in liposomes, peptide
conjugates, dendrimers, and direct microinjection of the DNA into
nuclei.
[0277] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells, NS0
cells, SP2 cells, HeLa cells, baby hamster kidney (BHK), cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), and a number of other cell lines. Non-mammalian
cells, including but not limited to, bacterial (e.g., E. coli and
Streptomyces species), yeast (e.g., Schizosaccharomyces pombe,
Saccharomyces cerevisiae and Pichia pastoris), insect (e.g., Sf9
cells), and plants can also be used to express recombinant
antibodies.
[0278] Production of recombinant antibodies in Chinese hamster
ovary (CHO) cells is the most widely used mammalian expression
system, particularly for production of antibodies. The most
commonly used CHO expression system is based on the use of CHO
cells deficient in the production of endogenous dihydrofolate
reductase (DHFR) coupled with a DHFR gene amplification system.
These DHFR.sup.- CHO cells are transfected with either a single
plasmid containing both antibody genes and a functional DHFR gene
or two plasmids with the DHFR gene contained on a separate plasmid
from the antibody gene cassettes. In other embodiments, the DHFR
gene is on the plasmid that encodes either the heavy or light
chain.
[0279] Transfected cells are selected in increasing concentrations
of the drug methotrexate. Survival on high concentrations of
methotrexate (1 to 10 .mu.M) is associated with gene amplification
of the DHFR gene during integration into the host chromosome or
integration into active regions of the chromosome. During the DHFR
gene amplification step, the antibody genes are also coamplified
and integrated into the host chromosome.
[0280] The expression methods are selected by determining which
system generates the highest expression levels and produce
antibodies with constitutive M-CSF binding properties. Further,
expression of antibodies of the invention (or other moieties
therefrom) from production cell lines can be enhanced using a
number of known techniques. For example, the glutamine synthetase
and DHFR gene expression systems are common approaches for
enhancing expression under certain conditions. High expressing cell
clones can be identified using conventional techniques, such as
limited dilution cloning and Microdrop technology. The Glutamine
Synthetase system is discussed in whole or part in connection with
European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and
European Patent Application No. 89303964.4.
[0281] In connection with the transgenic production in mammals,
antibodies can also be produced in, and recovered from, the milk of
goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,
5,756,687, 5,750,172, and 5,741,957.
[0282] When recombinant expression vectors encoding anti-M-CSF
antibody genes are introduced into host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. The antibodies
may be present in, the culture medium, whole cells, in a cell
lysate, or in a partially purified or substantially pure form. The
antibodies expressed in cell lines as described above may be
purified and/or isolated from the associated cellular material.
Purification is performed in order to eliminate other cellular
components or other contaminants, e.g. other cellular nucleic acids
or proteins, by standard techniques, including alkaline/SDS
treatment, column chromatography and others well known in the art.
See Ausubel, F., et al., ed. Current Protocols in Molecular
Biology, Greene Publishing and Wiley Interscience, New York (1987).
In one embodiment, the antibodies can be recovered from the culture
medium using protein purification methods, including the
purification methods described in the Examples herein.
[0283] In the present invention, it is possible that anti-M-CSF
antibodies expressed by different cell lines or in transgenic
animals will have different glycosylation patterns from each other.
However, all of the anti-M-CSF antibodies encoded by the nucleic
acids and amino acids provided herein are considered part of the
instant invention, regardless of their glycosylation pattern or
modification or deletion thereof.
[0284] As used herein, the term "glycosylation" means the pattern
of carbohydrate units that are covalently attached to an antibody.
When it is said that the anti-M-CSF antibodies herein have a
particular glycosylation pattern, it is meant that the majority of
the referenced anti-M-CSF antibodies have that particular
glycosylation pattern. In other aspects, when it is said that the
anti-M-CSF antibodies herein have a particular glycosylation
pattern, it is meant that greater than or equal to 75%, 90%, 95%,
or 99% of the referenced anti-M-CSF antibodies have that particular
glycosylation pattern. When the anti-M-CSF antibodies are
glycosylated they may have any possible glycosylation pattern.
Moreover, each heavy chain within one antibody may have the same
glycosylation pattern or the two heavy chains may have differing
glycosylation patterns.
[0285] The anti-M-CSF antibodies of the present invention also
encompass glycosylation variants of anti-M-CSF antibody 8.10.3F
(e.g., by insertion of a glycosylation site or deletion of any
glycosylation site by deletion, insertion or substitution of
suitable amino acid residues).
[0286] Glycosylation of polypeptides is typically either N-linked
or O-linked. Glycosylation of antibody polypeptides is typically
N-linked and forms a biantennary structure. N-linked refers to the
attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tri-peptide sequences asparagine-X-serine
and asparagine-X-threonine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tri-peptide sequences in an antibody
creates a potential glycosylation site.
[0287] The three distinct structures of biantennary glycans are
designated "G0", "G1" and "G2" having zero, one, or two,
respectively, terminal galactose residues on the nonreducing end of
the glycan. See Jefferis et al., Biochem. J., 268, 529-537 (1990).
In some cases, the glycan structure may also have a fucose residue
linked to an N-acetylglucosamine, which is covalently bonded to the
asparagine amino acid (e.g., position 297) found in the antibody.
When the fucose (F) is present, the biantennary glycan nomenclature
is changed to "G0F", "G1F", or "G2F" depending upon the number of
terminal galactose residues. See Teillaud, Expert Opin. Biol.
Ther., 5(Suppl. 1):S1327 (2005). Furthermore, when the antibody
contains both of the two heavy chains, the glycan nomenclature is
repeated for each of the two heavy chains.
[0288] For example, in one embodiment, the anti-M-CSF antibody
8.10.3F described herein has a glycosylation pattern of "G0F,G0F"
as reported in Example 10. The "G0F,G0F" glycoform is a species in
which both heavy chains have the G0 glycan attached and each G0
glycan has a fucose (F) residue linked to an N-acetylglucosamine,
which is covalently bonded to an asparagine amino acid at residue
297 found in the heavy chains of antibody 8.10.3F.
[0289] For purposes of the present invention, the anti-M-CSF
antibodies may be glycosylated or non-glycosylated. In certain
embodiments, the anti-M-CSF antibodies have a glycosylation pattern
that is selected from the group consisting of "G0F,G0F"; "G0F,G1F";
"G1F,G1F"; "G1F,G2F"; and mixtures thereof. In other embodiments,
the anti-M-CSF antibodies have a glycosylation pattern that is
"G0F,G0F". In still other embodiments, the anti-M-CSF antibodies
are not glycosylated.
[0290] Site directed mutagenesis of the antibody CH2 domain to
eliminate glycosylation is encompassed by the present invention in
order to prevent changes in either the immunogenicity,
pharmacokinetic, and/or effector functions resulting from non-human
glycosylation.
Routes of Administration and Dosages:
[0291] The compositions of this invention may be in liquid
solutions (e.g., injectable and infusible solutions). The preferred
form depends on the intended mode of administration and therapeutic
application. Typical preferred compositions are in the form of
injectable or infusible solutions, such as compositions similar to
those used for passive immunization of humans. The preferred mode
of administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular, and intrasternally) or by infusion
techniques, in the form of sterile injectable aqueous, liquid or
olagenous suspensions. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In a preferred embodiment, the
antibody is administered by intravenous infusion or injection. In
another preferred embodiment, the antibody is administered by
intramuscular or subcutaneous injection. Therapeutic compositions
typically are sterile and stable under the conditions of
manufacture and storage.
[0292] The composition can be formulated as a solution,
microemulsion, dispersion, or liposome. Sterile injectable
solutions can be prepared by incorporating the anti-M-CSF antibody
in the required amount in an appropriate diluent with one or a
combination of ingredients enumerated above, as required, followed
by sterilization (e.g., filter sterilization). Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. Such
suspensions may be formulated according to the known art using
those suitable dispersing of wetting agents and suspending agents
or other acceptable agents. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or diglycerides. In addition, n-3 polyunsaturated
fatty acids may find use in the preparation of injectables.
[0293] In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants.
[0294] Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin or by
formulating the composition into prolonged absorption forms such
as, depots, liposomes, polymeric microspheres, polymeric gels, and
implants.
[0295] Other methods for administration of the antibodies described
herein include dermal patches that release the medications directly
into a subject's skin. Such patches can contain the antibodies of
the present invention in an optionally buffered, liquid solution,
dissolved and/or dispersed in an adhesive, or dispersed in a
polymer.
[0296] Still other methods for administration of the antibodies
described herein include liquid opthalmological drops for the eyes,
including, without limitation, such ocular deliveries, as
intravitreal, subtenon, and intraocular.
[0297] The antibody may be administered once, but more preferably
is administered multiple times. For example, the antibody may be
administered from once daily to once every six months or longer.
The administering may be on a schedule such as three times daily,
twice daily, once daily, once every two days, once every three
days, once weekly, once every two weeks, once every month, once
every two months, once every three months and once every six
months.
[0298] The antibody may also be administered continuously via a
minipump. The antibody may be administered at the site of a tumor
or inflamed body part, into the tumor or inflamed body part or at a
site distant from the site of the tumor or inflamed body part. The
antibody may be administered once, at least twice or for at least
the period of time until the condition is treated, palliated or
cured. The antibody generally may be administered for as long as
the tumor or inflammation is present provided that the antibody
causes the tumor or cancer to stop growing or to decrease in weight
or volume or until the inflamed body part experiences a reduction
in inflammation. The antibody typically would be administered as
part of a pharmaceutical composition as described supra.
[0299] The compositions of the invention may include a
therapeutically effective amount or a prophylactically effective
amount of an antibody or antigen-binding portion of the invention.
In preparing the composition, the therapeutically effective amount
of the anti-M-CSF antibody present in the composition can be
determined, for example, by taking into account the desired dose
volumes and mode(s) of administration, the nature and severity of
the condition to be treated, and the age and size of the
subject.
[0300] Exemplary, non-limiting dose ranges for administration of
the pharmaceutical compositions of the present invention to a
subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in
terms of milligrams (mg) of anti-M-CSF antibody administered per
kilogram (kg) of subject weight), from about 0.01 mg/kg to about
100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1
mg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 3 mg/kg
For purposes of the present invention, an average human subject
weighs about 70 kg. In addition, the quantity of active component
in a unit dose preparation may be varied or adjusted from 0.1 mg to
100 mg and from 0.5 mg to 100 mg, according to the particular
application and the potency of the active component. Ranges
intermediate to any of the dosages cited herein, e.g., about 0.01
mg/kg-199 mg/kg, are also intended to be part of this invention.
For example, ranges of values using a combination of any of the
recited values as upper and/or lower limits are intended to be
included.
[0301] Dosage regimens can also be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response) by
administering several divided doses to a subject over time or the
dose can be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage.
[0302] Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the anti-M-CSF antibody or portion and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
antibody for the treatment of sensitivity in individuals.
[0303] The liquid compositions of the present invention can be
prepared as unit dosage forms. For example, a unit dosage per vial
may contain from 1 to 1000 milliliters (mls) of different
concentrations of an anti-M-CSF antibody. In other embodiments, a
unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5
ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50
ml or 100 ml of different concentrations of an anti-M-CSF antibody.
If necessary, these preparations can be adjusted to a desired
concentration by adding a sterile diluent to each vial.
[0304] The liquid compositions of the present invention can also be
prepared as unit dosage forms in sterile bags or containers, which
are suitable for connection to an intravenous administration line
or catheter.
Stability Assessment:
[0305] The present invention comprises stable compositions
comprising an anti-M-CSF antibody as described herein and a
chelating agent. A stable composition is desirable to maintain or
resist changes in, for example, product appearance and integrity
(including physical or chemical degradation potentially leading to
a reduction in biological activity). Various analytical techniques
and indicators for measuring protein stability are reported in the
literature and a number of these techniques and indicators 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). In general, the liquid
pharmaceutical compositions of the present invention exhibit
improved stability when subjected to low storage temperatures over
a period of time.
[0306] In one embodiment, the composition when stored at a
temperature ranging from about 2.degree. C. to about 8.degree. C.
(preferably, about 5.degree. C.) for at least about 12 months,
preferably at least about 18 months and more preferably at least
about 24 months, is more stable than an isotonic composition
lacking the chelating agent that is stored under the same
conditions for the same time.
[0307] In another embodiment, the composition when stored at a
temperature from about 25.degree. C. to about 30.degree. C. for at
least about 3 months, preferably at least 6 months, and more
preferably at least about 12 months, is more stable than an
isotonic composition lacking the chelating agent that is stored
under the same conditions for the same time.
[0308] In another embodiment, the composition when stored at a
temperature of about 40.degree. C. for at least about 1 months,
preferably at least about 2 months, more preferably at least about
3 months, and yet more preferably at least about 26 weeks, is more
stable than an isotonic composition lacking the chelating agent
that is stored under the same conditions for the same time.
[0309] As used herein, the term "a freeze/thaw cycle" refers to
techniques for using a liquid antibody sample after frozen storage,
wherein the temperature of the sample is lowered to a temperature
of 0.degree. C. or lower in order to freeze the liquid sample, and
then subjecting the sample to a temperature which will restore its
liquid state for a sufficient period of time to permit use of the
sample, followed by and return to frozen storage, preferably at a
temperature of 0.degree. C. or lower. As used herein, the term
"frozen storage" refers to freezing and maintaining a previously
liquid antibody sample at a temperature of 0.degree. C. or below,
and preferably -20.degree. C. or lower.
[0310] In one embodiment, the composition when subjected to at
least 1 freeze/thaw cycle, preferably at least 2 freeze/thaw
cycles, more preferably at least 3 freeze/thaw cycles, still more
preferably at least 4 freeze/thaw cycles, still more preferably at
least 5 freeze/thaw cycles, and still more preferably at least 6
freeze/thaw cycles, remains stable.
[0311] In another embodiment, the composition satisfies two or more
of the following conditions:
[0312] (a) the composition when stored at a temperature from about
2.degree. C. to about 8.degree. C. for at least about 12 months,
preferably at least about 18 months and more preferably at least
about 24 months, is more stable than an isotonic composition
lacking the chelating agent that is stored under the same
conditions for the same time;
[0313] (b) the composition when stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months, is more stable than an isotonic composition lacking the
chelating agent that is stored under the same conditions for the
same time;
[0314] (c) the composition when stored at a temperature of about
40.degree. C. for at least about 1 months, preferably at least
about 2 months, more preferably at least about 3 months, and yet
more preferably at least about 26 weeks, is more stable than an
isotonic composition lacking the chelating agent that is stored
under the same conditions for the same time; or
[0315] (d) the composition when subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw cycles,
remains stable.
[0316] In another embodiment, the composition satisfies three or
more of the conditions discussed immediately above.
[0317] For purposes of the present application, antibody
aggregation, antibody fragmentation, and/or composition
discoloration, for example, can be used as indicators of the
stability of the composition. In general, the compositions of the
present invention exhibit a lower level of at least one of antibody
aggregation, antibody fragmentation and composition discoloration
when subjected to one or more of the above-described storage or
freeze/thaw conditions relative to isotonic compositions lacking
the chelating agent that are subjected to the same conditions.
[0318] Protein aggregation in a liquid pharmaceutical composition
can be measured by various methods known in the art. Such methods
include gel filtration chromatography to separate proteins on the
basis of their molecular weight and/or size. A "gel" is a matrix of
water and a polymer, such as agarose or polymerized acrylamide. The
present invention also encompasses the use of gel filtration HPLC
(high performance liquid chromatography) (also known as
Size-Exclusion HPLC, i.e., SE-HPLC). Other recognized methods of
measuring aggregation include cation exchange chromatography, which
is the general liquid chromatographic technique of ion-exchange
chromatography utilizing anion columns. The cations exchanged in
the present invention are from the protein molecules. Since
multivalent protein aggregates may have some multiple of the net
charge of the antigen-binding monomer protein, the aggregates can
be retained more strongly, and may be separated from the monomer
molecules. A preferred cationic exchanger is a polyaspartic acid
column. Thus, a monomeric protein can be readily distinguished from
an aggregate. However, those of ordinary skill in the art will
realize that aggregation assays of the invention are not limited to
any particular type of chromatography column, so long as it is
capable of separating the two forms of protein molecules.
[0319] Protein fragmentation in a composition of the present
invention can be measured by various methods known in the art. Such
methods include, for example, size exclusion chromatography,
ultraviolet detection (e.g., at 214 nanometers), SDS-PAGE and/or
matrix-assisted laser desorption ionization/time-of-flight mass
spectrometry (MALDI/TOF MS). Protein fragmentation resulting in a
charge alteration (e.g., occurring as a result of deamidation) can
be evaluated, for example, by ion-exchange chromatography or
isoelectric focusing (IEF).
[0320] Composition discoloration generally can be measured by
visual observation of the composition itself. The present liquid
pharmaceutical compositions comprising a chelating agent generally
reduce composition discoloration (e.g., pink or yellow) relative to
isotonic compositions that do not contain the chelating agent. For
purposes of the present invention, the term "discoloration" refers
to both changes in color (e.g., from clear and colorless to pink or
yellow) and to changes in clarity (e.g., from clear and colorless
to turbid, cloudy and/or having particulates). Composition
discoloration generally can be measured using additional techniques
such as by spectrophotometric (ultraviolet or visible wavelength
detection, e.g., at 214 nanometers), fluorimetric detection and/or
by visual comparison against a standard color scale of the
compositions with and without the chelating agent. See PhEur 5.0,
2005 Monograph 2.2.2.
[0321] In one embodiment, antibody aggregation is determined after
the composition is subjected to at least one of the following
conditions:
[0322] (a) the composition is stored at a temperature ranging from
about 2.degree. C. to about 8.degree. C. (preferably, about
5.degree. C.) for at least about 12 months, preferably at least
about 18 months and more preferably at least about 24 months;
[0323] (b) the composition is stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months;
[0324] (c) the composition is stored at a temperature of about
40.degree. C. for at least about 1 months, preferably at least
about 2 months, more preferably at least about 3 months, and yet
more preferably at least about 26 weeks; or
[0325] (d) the composition is subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw cycles.
Antibody aggregates are then chromatographically separated (e.g.,
using HPLC) and the extent of aggregation determined from the
resulting chromatogram.
[0326] In one embodiment, the present invention also provides a
composition comprising at least one antibody wherein the antibody
comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2
and a light chain amino acid sequence comprising SEQ ID NO: 4,
wherein the antibody binds to human M-CSF; and a chelating agent,
and the antibody is stable at a temperature of about 5.degree. C.
for at least about 26 weeks.
[0327] In another embodiment, the present invention also provides a
composition comprising at least one antibody wherein the antibody
comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2
and a light chain amino acid sequence comprising SEQ ID NO: 4,
wherein the antibody binds to human M-CSF; and a chelating agent,
and the antibody is stable at a temperature of about 25.degree. C.
for at least about 26 weeks.
[0328] In another embodiment, the present invention also provides a
composition comprising at least one antibody wherein the antibody
comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2
and a light chain amino acid sequence comprising SEQ ID NO: 4,
wherein the antibody binds to human M-CSF; and a chelating agent,
and the antibody is stable at a temperature of about 40.degree. C.
for at least about 26 weeks.
[0329] The stable compositions of the present invention typically
have an aggregate peak area on the chromatogram that is less than
or equal to any of the following: about 8.0%, about 7.5%, about
7.0%, about 6.5%, about 6.0%, about 5.5%, about 5.0%, about 4.5%,
about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%,
about 1.0%, about 0.9%, or about 0.8% of the total peak area on the
chromatogram. In one specific example of this technique for
measuring aggregation, the composition is stored for 26 weeks at
40.degree. C. and chromatographic separation is then conducted
using SE-HPLC with ultraviolet detection at 214 nanometers. This
technique was used to measure antibody aggregation in Example 9
where, for example, Formulation 11 (containing a chelating agent)
exhibited an aggregate peak area on the chromatogram of about 4.9%
while Formulation 9 (isotonic, but lacking a chelating agent)
exhibited an aggregate peak area on the chromatogram of about
11.6%.
[0330] In general, the difference between the aggregate
chromatogram peak area for a stable composition of the present
invention and the aggregate chromatogram peak area for an isotonic
composition lacking the chelating agent that is subjected to the
same conditions is at least about 8.0% or greater, is least about
7.5% or greater, is least about 7.0% or greater, is at least about
6.5% or greater, is at least about 6.0% or greater, is at least
about 5.5% or greater, at least about 5.0% or greater, at least
about 4.5% or greater, at least about 4% or greater, at least about
3.5% or greater, at least about 3.0% or greater, at least about
2.5% or greater, at least about 2.0% or greater, at least about
1.5% or greater, at least about 1.0% or greater, at least about
0.5% or greater, at least about 0.3% or greater, at least about
0.2% or greater, or at least about 0.1% or greater. For example,
the difference between Formulation 11 (aggregate peak area on the
chromatogram of about 4.9%) and Formulation 9 (aggregate peak area
on the chromatogram of about 11.6%) tested in Example 9 as
discussed above is about 6.7%.
[0331] In another embodiment, antibody fragmentation is determined
after the composition is subjected to at least one of the following
conditions:
[0332] (a) the composition is stored at a temperature ranging from
about 2.degree. C. to about 8.degree. C., (preferably, about
5.degree. C.) for at least about 12 months, preferably at least
about 18 months and more preferably at least about 24 months;
[0333] (b) the composition is stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months;
[0334] (c) the composition is stored at a temperature of about
40.degree. C. for at least about 1 months, preferably at least
about 2 months, more preferably at least about 3 months, and yet
more preferably at least about 26 weeks; or
[0335] (d) the composition is subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw
cycles.
[0336] Without wishing to be bound by theory it is believed that
one of the anti-M-CSF antibody 8.10.3F fragments is the result of
cleavage of the peptide bond between Asp99 and Pro100 within the
heavy chain variable domain (V.sub.H) amino acid sequence of SEQ ID
NO: 2 for antibody 8.10.3F. In certain embodiments, the
aforementioned antibody fragment results in a fragment that ranges
in size (i.e., weight) of from about 10.5 kD to about 11.5 kD, and
in some embodiments, the band is about 11 kD, and in some
embodiments, the band is about 10.8 kD. A reducing SDS-PAGE gel,
run with appropriate size markers, can be used to visualize
antibody fragment banding patterns and their sizes.
[0337] In general, the difference between the fragment band volume
for a stable composition of the present invention and the fragment
band volume for an isotonic composition lacking the chelating agent
that is subjected to the same conditions is at least about 8.0% or
greater, is least about 7.5% or greater, is least about 7.0% or
greater, is at least about 6.5% or greater, is at least about 6.0%
or greater, is at least about 5.5% or greater, at least about 5.0%
or greater, at least about 4.5% or greater, at least about 4% or
greater, at least about 3.5% or greater, at least about 3.0% or
greater, at least about 2.5% or greater, at least about 2.0% or
greater, at least about 1.5% or greater, at least about 1.0% or
greater, at least about 0.5% or greater, at least about 0.3% or
greater, at least about 0.2% or greater, or at least about 0.1% or
greater. For example, the difference between Formulation 11
(fragment band volume on the chromatogram of about 1.7%) and
Formulation 9 (fragment band volume on the chromatogram of about
3.5%) tested in Example 9 as discussed above is about 1.8%.
[0338] In one embodiment, the present invention also provides a
stable composition comprising at least one monoclonal anti-M-CSF
antibody and a stabilizing amount of a chelating agent, wherein
after the composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between a fragment
chromatogram peak area for the stable composition comprising at
least one monoclonal anti-M-CSF antibody and the chelating agent,
and a fragment chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 1%; or the decrease
between a fragment chromatogram peak area for the stable
composition comprising at least one monoclonal anti-M-CSF antibody
and the chelating agent, and a fragment chromatogram peak area for
an isotonic composition lacking the chelating agent, is at least
about 0.5%.
[0339] In another embodiment, the present invention also provides a
stable composition comprising at least one monoclonal anti-M-CSF
antibody and a stabilizing amount of a chelating agent, wherein
after the composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between a fragment
chromatogram peak area for the stable composition comprising at
least one monoclonal anti-M-CSF antibody and the chelating agent,
and a fragment chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 3.5%; or the
decrease between a fragment chromatogram peak area for the stable
composition comprising at least one monoclonal anti-M-CSF antibody
and the chelating agent, and a fragment chromatogram peak area for
an isotonic composition lacking the chelating agent, is at least
about 1.7%.
[0340] In another embodiment, the present invention also provides a
method for stabilizing at least one monoclonal anti-M-CSF antibody
comprising the method of forming a composition comprising the
antibodies and a stabilizing amount of a chelating agent, wherein
after the composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between an aggregate
chromatogram peak area for the stable composition comprising
monoclonal anti-M-CSF antibodies and the chelating agent, and an
aggregate chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 1%; or the decrease
between an aggregate chromatogram peak area for the stable
composition comprising monoclonal anti-M-CSF antibodies and the
chelating agent, and an aggregate chromatogram peak area for an
isotonic composition lacking the chelating agent, is at least about
0.5%.
[0341] In another embodiment, the present invention also provides a
method for stabilizing at least one monoclonal anti-M-CSF antibody
comprising the method of forming a composition comprising the
antibodies and a stabilizing amount of a chelating agent, wherein
after the composition is stored for a period of about 26 weeks at a
temperature of about 40.degree. C., one or both of the following
conditions are satisfied: the decrease between an aggregate
chromatogram peak area for the stable composition comprising
monoclonal anti-M-CSF antibodies and the chelating agent, and an
aggregate chromatogram peak area for an isotonic composition
lacking the chelating agent, is at least about 11.6%; and/or the
decrease between an aggregate chromatogram peak area for the stable
composition comprising monoclonal anti-M-CSF antibodies and the
chelating agent, and a aggregate chromatogram peak area for an
isotonic composition lacking the chelating agent, is at least about
4.9%.
[0342] In another embodiment, the present invention also provides
methods for stabilizing at least one monoclonal anti-M-CSF antibody
in a composition comprising the method: forming a composition
comprising the antibody and at least one stabilizing chelating
agent compound, wherein the composition comprises a sufficient
amount of the stabilizing chelating agent to stabilize the
composition during storage for a period of about 26 weeks at a
temperature of about 40.degree. C.; and wherein at the end of the
storage period at least one of the following conditions are
satisfied: the amount of aggregated antibodies is less than or
equal to about 3.5% by peak area of a chromatogram of the
antibodies after chromatographic separation; or the amount of
antibody fragment formed by antibody hydrolysis having a molecular
weight ranging from about 10.5 kD to about 11.5 kD is less than or
equal to about 1.7% by peak area of a chromatogram of the
antibodies after chromatographic separation.
[0343] In another embodiment, the present invention also provides
methods for stabilizing at least one monoclonal anti-M-CSF antibody
8.10.3F in a composition comprising the method: forming a
composition comprising the antibody and at least one stabilizing
chelating agent compound, wherein the composition comprises a
sufficient amount of the stabilizing chelating agent to stabilize
the composition during storage for a period of about 26 weeks at a
temperature of about 40.degree. C.; and wherein at the end of the
storage period at least one of the following conditions are
satisfied: the amount of aggregated antibodies is less than or
equal to about 3.5% by peak area of a chromatogram of the
antibodies after chromatographic separation; or the amount of
antibody fragment formed by antibody hydrolysis having a molecular
weight ranging from about 10.5 kD to about 11.5 kD is less than or
equal to about 1.7% by peak area of a chromatogram of the
antibodies after chromatographic separation.
[0344] Accordingly, the present invention provides a method for
stabilizing at least one anti-M-CSF antibody by combining the
antibody in a liquid composition with a chelating agent in an
amount, which reduces chemical or physical instability of the
antibody.
[0345] Likewise, the present invention provides a method for
analyzing the stability of at least one anti-M-CSF antibody
comprising identifying an antibody fragment formed by antibody
hydrolysis having a molecular weight ranging from about 10.5 kD to
about 11.5 kD polypeptide fragment in a composition comprising the
anti-M-CSF antibody by separating species by organic SE-HPLC, and
identifying the presence of the polypeptide fragment in the
composition by using ultraviolet detection at 214 nanometers.
[0346] Also provided is a method for detecting the presence of a
polypeptide fragment having a molecular weight of between about 10
kD and about 12 kD in a composition comprising at least one
anti-M-CSF antibody antibody, the method comprising separating
species by organic SE-HPLC, and identifying the presence of the
polypeptide fragment in the composition by using ultraviolet
detection at 214 nanometers or at other suitable wavelengths, e.g.,
280 nm.
Methods of Treatment:
[0347] Any of the types of antibodies described herein may be used
therapeutically. In a preferred embodiment, the anti-M-CSF antibody
is a human antibody. In another preferred embodiment, the M-CSF is
human M-CSF and the subject is a human subject. In yet another
preferred embodiment, the anti-M-CSF antibody is a human IgG2
antibody. Alternatively, the subject may be a mammal that expresses
an M-CSF protein that the anti-M-CSF antibody cross-reacts with.
The antibody may be administered to a non-human mammal expressing
M-CSF with which the antibody cross-reacts (i.e., a primate) for
veterinary purposes or as an animal model of human disease. Such
animal models may be useful for evaluating the therapeutic efficacy
of antibodies of this invention.
[0348] In one embodiment, the present invention provides a method
for the treatment of an M-CSF-mediated disorder in a subject,
comprising administering to the subject a therapeutically effective
amount of a liquid pharmaceutical composition comprising: at least
one antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a
pharmaceutically acceptable excipient.
[0349] In one embodiment, the present invention provides a method
for the treatment of an inflammatory disease in a subject,
comprising administering to the subject a therapeutically effective
amount of a liquid pharmaceutical composition comprising: at least
one antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a
pharmaceutically acceptable excipient comprising a chelating agent
alone or in combination with other excipients chosen from a buffer,
antioxidant, a tonicity agent, or a surfactant, and mixtures
thereof. In further embodiments, the aforementioned subject is one
that is in need of the treatment of an inflammatory disease. In
other embodiments, the methods and compositions of the present
invention encompass the treatment of the inflammatory diseases
selected from the group consisting of pain, fever, inflammation,
atherosclerosis, sepsis, asthma, autoimmune diseases, osteoporosis,
rheumatoid arthritis, and osteoarthritis.
[0350] In another embodiment, the present invention provides a
method for the treatment of a neoplasia disorder in a subject,
comprising administering to the subject a therapeutically effective
amount of a liquid pharmaceutical composition comprising: at least
one antibody comprising an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, and further comprising an amino acid sequence that is at least
90% identical to a heavy chain amino acid sequence shown in SEQ ID
NO: 2, wherein the antibody binds to human M-CSF; and a
pharmaceutically acceptable excipient comprising a chelating agent
alone or in combination with other excipients chosen from a buffer,
an antioxidant, a tonicity agent, or a surfactant, and mixtures
thereof. In further embodiments, the aforementioned subject is one
that is in need of the treatment of a neoplasia disorder.
[0351] Both of the terms, "neoplasia" and "neoplasia disorder",
refer to a "neoplasm" or tumor, which may be benign, premalignant,
metastatic, or malignant. Also encompassed by the present invention
are benign, premalignant, metastatic, or malignant neoplasias. Also
encompassed by the present invention are benign, premalignant,
metastatic, or malignant tumors. Thus, all of benign, premalignant,
metastatic, or malignant neoplasia or tumors are encompassed by the
present invention and may be referred to interchangeably, as
neoplasia, neoplasms or neoplasia-related conditions. Tumors are
generally known in the art to be a mass of neoplasia or
"neoplastic" cells. Although, it is to be understood that even one
neoplastic cell is considered, for purposes of the present
invention to be a neoplasm or alternatively, neoplasia.
[0352] Neoplasia disorders that may be treated by an anti-M-CSF
antibody of the invention can involve any tissue or organ, and
include, but are not limited to bone, brain, lung, squamous cell,
bladder, gastric, pancreatic, breast, head, neck, liver, renal,
ovarian, prostate, colorectal, esophageal, gynecological (e.g.,
cervical and ovarian), nasopharynx, or thyroid cancers. Also
encompassed by the term neoplasia disorders, are bone metastases,
melanomas, lymphomas, leukemias, and multiple myelomas. In
particular, the anti-M-CSF antibody formulations of the present
invention are useful to treat cancers of the breast, prostate,
colon and lung.
[0353] In other embodiments, the methods and compositions of the
present invention encompass the prevention and treatment of the
neoplasia disorders selected from the group consisting of acral
lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid
cycstic carcinoma, adenomas, familial adenomatous polyposis,
familial polyps, colon polyps, polyps, adenosarcoma, adenosquamous
carcinoma, adrenocortical carcinoma, AIDS-related lymphoma, anal
cancer, astrocytic tumors, bartholin gland carcinoma, basal cell
carcinoma, bile duct cancer, bladder cancer, brain stem glioma,
brain tumors, breast cancer, bronchial gland carcinomas, capillary
carcinoma, carcinoids, carcinoma, carcinoma of the fallopian tubes,
carcinoma of the endometrium, carcinosarcoma, cavernous, central
nervous system lymphoma, cerebral astrocytoma, cholangiocarcinoma,
chondosarcoma, choriod plexus papilloma/carcinoma, clear cell
carcinoma, skin cancer, brain cancer, colon cancer, colorectal
cancer, cutaneous T-cell lymphoma, cystadenoma, endodermal sinus
tumor, endometrial hyperplasia, endometrial stromal sarcoma,
endometrioid adenocarcinoma, ependymal, epitheloid, esophageal
cancer, Ewing's sarcoma, extragonadal germ cell tumor,
fibrolamellar, focal nodular hyperplasia, gallbladder cancer,
gastrinoma, germ cell tumors, gestational trophoblastic tumor,
glioblastoma, glioma, glucagonoma, hemangiblastomas,
hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic
adenomatosis, hepatocellular carcinoma, Hodgkin's lymphoma,
hypopharyngeal cancer, hypothalamic and visual pathway glioma,
insulinoma, intaepithelial neoplasia, interepithelial squamous cell
neoplasia, intraocular melanoma, invasive squamous cell carcinoma,
large cell carcinoma, islet cell carcinoma, Kaposi's sarcoma,
kidney cancer, laryngeal cancer, leiomyosarcoma, lentigo maligna
melanomas, leukemia-related conditions, lip and oral cavity cancer,
liver cancer, lung cancer, lymphoma, malignant mesothelial tumors,
malignant thymoma, medulloblastoma, medulloepithelioma, melanoma,
meningeal, merkel cell carcinoma, mesothelial, metastatic
carcinoma, mucoepidermoid carcinoma, multiple myeloma/plasma cell
neoplasm, mycosis fungoides, myelodysplastic syndrome,
myeloproliferative conditions, nasal cavity and paranasal sinus
cancer, nasopharyngeal cancer, neuroblastoma, neuroepithelial
adenocarcinoma nodular melanoma, neoplasms of the central nervous
system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem
gliomas or pituitary adenomas), non-Hodgkin's lymphoma, oat cell
carcinoma, oligodendroglial, oral cancer, oropharyngeal cancer,
osteosarcoma, pancreatic polypeptide, ovarian cancer, ovarian germ
cell tumor, pancreatic cancer, papillary serous adenocarcinoma,
pineal cell, pituitary tumors, plasmacytoma, pseudosarcoma,
pulmonary blastoma, parathyroid cancer, penile cancer,
pheochromocytoma, pineal and supratentorial primitive
neuroectodermal tumors, pituitary tumor, plasma cell neoplasm,
pleuropulmonary blastoma, prostate cancer, rectal cancer, renal
cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous
carcinoma, small cell carcinoma, small intestine cancer, soft
tissue carcinomas, somatostatin-secreting tumor, squamous
carcinoma, squamous cell carcinoma, submesothelial, superficial
spreading melanoma, supratentorial primitive neuroectodermal
tumors, thyroid cancer, undifferentiatied carcinoma, urethral
cancer, uterine cancer, uveal melanoma, verrucous carcinoma,
vaginal cancer, vipoma, vulvar cancer, Waldenstrom's
macroglobulinemia, well differentiated carcinoma, and Wilm's
tumor.
[0354] In a more preferred embodiment, the anti-M-CSF antibody is
administered to a subject with breast cancer, prostate cancer, lung
cancer or colon cancer. In an even more preferred embodiment, the
method causes the cancer to stop proliferating abnormally, or not
to increase in weight or volume or to decrease in weight or
volume.
[0355] The compositions of the present invention may be used in
combination with agents useful for treating a cancer in a mammal
such as chemotherapeutic agents. In some embodiments, the
chemotherapeutic agent is selected from the group consisting of
mitotic inhibitors, alkylating agents, anti-metabolites,
intercalating antibiotics, growth factor inhibitors, cell cycle
inhibitors, enzymes, topoisomerase inhibitors, biological response
modifiers, tamoxifen, anti-hormones, e.g., anti-androgens, and
anti-angiogenesis agents.
[0356] In addition, a composition of a human anti-M-CSF monoclonal
antibody of the invention can also be used with signal transduction
inhibitors, such as agents that can inhibit EGF-R (epidermal growth
factor receptor) responses, such as EGF-R antibodies, EGF
antibodies, and molecules that are EGF-R inhibitors; VEGF (vascular
endothelial growth factor) inhibitors, such as VEGF receptors and
molecules that can inhibit VEGF; and erbB2 receptor inhibitors,
such as organic molecules or antibodies that bind to the erbB2
receptor, for example, HERCEPTIN.TM. (Genentech, Inc.).
EGFR-inhibiting agents include, but are not limited to, the
monoclonal antibodies C225 and anti-EGFR 22 Mab (ImClone Systems
Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck
KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck
KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839
(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis),
PTK 787 (Novartis), CP 701 (Cephalon), leflunomide
(Pharmacia/Sugen), CI-1033 (Warner Lambert Parke Davis), CI-1033/PD
183, 805 (Warner Lambert Parke Davis), CL-387,785 (Wyeth-Ayerst),
BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol
Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer
Ingelheim), OLX-103 (Merck & Co.), VRCTC-310 (Ventech
Research), EGF fusion toxin (Seragen Inc.), DAB-389
(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund),
RG-50864 (INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97
(Parker Hughes Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa
Hakko) and EGF-R Vaccine (York Medical/Centro de Immunologia
Molecular (CIM)). These and other EGF-R-inhibiting agents can be
used in the present invention.
[0357] VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen,
Inc.), AVASTIN.TM. (Genentech), SH-268 (Schering), and NX-1838
(NeXstar) can also be combined with the compound of the present
invention. Anti-inflammatory agents can be used in conjunction with
an anti-M-CSF antibody formulation of the present invention. For
the treatment of inflammatory diseases such as rheumatoid
arthritis, the human anti-M-CSF antibodies of the invention may be
combined with agents such as TNF-.alpha. inhibitors such as TNF
drugs (such as REMICADE.TM., CDP-870 and HUMIRA.TM.) and TNF
receptor immunoglobulin molecules (such as ENBREL.TM.), CTLA-4 lg,
anti-CD20 antibodies (e.g., rituxamab), IL-6 antibodies, IL-6
receptor antibodies e.g., tocilizumab), IL-1 inhibitors, IL-1
receptor antagonists or soluble IL-1ra (e.g. Kineret or ICE
inhibitors), Cox-2 inhibitors (such as celecoxib, rofecoxib,
valdecoxib and etoricoxib), metalloprotease inhibitors (preferably
MMP-13 selective inhibitors), p2X7 inhibitors, .alpha.2.delta.
ligands (such as NEURONTIN.TM. AND PREGABALIN.TM.), low dose
methotrexate, sulfasalazine, Mesalamine leflunomide,
hydroxychloroquine, d-penicillamine, auranofin or parenteral or
oral gold.
[0358] The compositions of the invention can also be used in
combination with existing therapeutic agents for the treatment of
osteoarthritis. Suitable agents to be used in combination include
standard non-steroidal anti-inflammatory agents (hereinafter
NSAID's) such as piroxicam, diclofenac, propionic acids such as
naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen,
fenamates such as mefenamic acid, indomethacin, sulindac, apazone,
pyrazolones such as phenylbutazone, salicylates such as aspirin,
Cox-2 inhibitors such as celecoxib, valdecoxib, rofecoxib and
etoricoxib, analgesics and intraarticular therapies such as
corticosteroids and hyaluronic acids such as hyalgan and
synvisc.
[0359] The human anti-M-CSF antibody compositions of the present
invention may also be used in combination with cardiovascular
agents such as calcium channel blockers, lipid lowering agents such
as statins (e.g., atorvastain calcium), fibrates, beta-blockers,
ACE inhibitors, Angiotensin-2 receptor antagonists, and platelet
aggregation inhibitors.
[0360] The compositions of the present invention may also be used
in combination with CNS agents such as antidepressants (such as
sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa,
REQUIP.TM., MIRAPEX.TM., MAOB inhibitors such as selegine and
rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors,
dopamine reuptake inhibitors, NMDA antagonists, nicotine agonists,
dopamine agonists and inhibitors of neuronal nitric oxide
synthase), and anti-Alzheimer's drugs such as donepezil, Tacrine,
.alpha.2.delta. Ligands (such NEUROTIN.TM. and PREGABALIN.TM.)
inhibitors, Cox-2 inhibitors, propentofylline or metrifonate.
[0361] The anti-M-CSF antibody compositions of the present
invention may also be used in combination with osteoporosis agents
such as roloxifene, droloxifene, lasofoxifene or fosomax and
immunosuppressant agents such as FK-506 and rapamycin.
Articles of Manufacture
[0362] In another embodiment of the invention, an article of
manufacture is provided comprising a container, which holds the
composition comprising at least one of the monoclonal anti-M-CSF
antibodies of the present invention in combination with a
pharmaceutically acceptable chelating agent, and optionally
provides instructions for its use. Suitable containers include, for
example, bottles, bags, vials and syringes. The container may be
formed from a variety of materials such as glass or plastic. An
exemplary container is a 3-20 cc single use glass vial.
Alternatively, for a multidose formulation, the container may be
3-100 cc glass vial. The container holds the formulation and the
label on, or associated with, the container may indicate directions
for use. 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, contraindications,
and/or lists of potential side-effects.
[0363] The present invention also provides a kit for preparing a
composition of an antibody comprising a first container comprising
monoclonal anti-M-CSF antibody 8.10.3F, and a second container
comprising a pharmaceutically acceptable chelating agent.
[0364] The following examples describe embodiments of the
invention. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the
invention being indicated by the claims, which follow the examples.
In the examples, all percentages are given on a weight basis unless
otherwise indicated. The skilled artisan will appreciate that the
weight quantities and/or weight-to-volume ratios recited in the
examples can be converted to moles and/or molarities using the
art-recognized molecular weights of the recited ingredients. Weight
quantities exemplified herein (e.g., grams) are for the volumes
(e.g., of buffer solutions, antibody formulation, etc.) recited.
The skilled artisan will appreciate that the weight quantities can
be proportionally adjusted when different formulation volumes are
desired.
EXAMPLE 1
[0365] This Example shows the generation of hybridoma cell lines
that produce anti-M-CSF antibodies as described in U.S. Published
Application No. 20050059113 to Bedian, et al.
Immunization and Hybridoma Generation
[0366] Eight to ten week old XENOMOUSE.TM. mice were immunized
intraperitoneally or in their hind footpads with human M-CSF (10
.mu.g/dose/mouse). This dose was repeated five to seven times over
a three to eight week period. Four days before fusion, the mice
were given a final injection of human M-CSF in phosphate buffered
saline (PBS). The spleen and lymph node lymphocytes from immunized
mice were fused with the non-secretory myeloma P3-X63-Ag8.653 cell
line, and the fused cells were subjected to HAT selection. See
Galfre, G. and Milstein, C., "Preparation of monoclonal antibodies:
strategies and procedures." Methods Enzymol. 73:3-46 (1981). A
panel of hybridomas all secreting M-CSF specific human IgG2 and
IgG4 antibodies was recovered. Antibodies also were generated using
XENOMAX.TM. technology as described in Babcook, J. S. et al., Proc.
Natl. Acad. Sci. USA 93:7843-48, 1996. Nine cell lines engineered
to produce antibodies of the invention were selected for further
study and designated 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4,
8.10.3 and 9.7.2. The hybridomas were deposited under terms in
accordance with the Budapest Treaty with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209
on Aug. 8, 2003. The hybridomas were assigned the following
accession numbers:
TABLE-US-00002 Hybridoma 3.8.3 (LN 15891) PTA-5390 Hybridoma 2.7.3
(LN 15892) PTA-5391 Hybridoma 1.120.1 (LN 15893) PTA-5392 Hybridoma
9.7.2 (LN 15894) PTA-5393 Hybridoma 9.14.4 (LN 15895) PTA-5394
Hybridoma 8.10.3 (LN 15896) PTA-5395 Hybridoma 88-gamma (UC 25489)
PTA-5396 Hybridoma 88-kappa (UC 25490) PTA-5397 Hybridoma 100-gamma
(UC 25491) PTA-5398 Hybridoma 100-kappa (UC 25492) PTA-5399
Hybridoma 252-gamma (UC 25493) PTA-5400 Hybridoma 252-kappa (UC
25494) PTA-5401
EXAMPLE 2
[0367] This Example shows the generation of a recombinant mammalian
cell line that produces anti-M-CSF antibodies.
[0368] DNA encoding the heavy and light chains of monoclonal
antibodies 8.10.3 was cloned from the respective hybridoma cell
line 8.10.3 and the DNA sequences were determined by methods known
to one skilled in the art. The DNA from the hybridoma cell line
8.10.3 was mutated at specific framework regions in the variable
domain to obtain 8.10.3F. From nucleic acid sequence and predicted
amino acid sequence of the antibody 8.10.3F, the identity of the
gene usage for each antibody chain was determined by ("VBASE").
Table 2 sets forth the gene utilization of antibody 8.10.3F in
accordance with the present invention:
TABLE-US-00003 TABLE 2 Heavy and Light Chain Human Gene Utilization
and Sequences Heavy Chain Light Chain Antibody SEQ ID NO: V.sub.H
D.sub.H J.sub.H SEQ ID NO: V.sub.K J.sub.K 8.10.3F 1 (nucleic 3-48
1-26 4b 3 (nucleic A27 4 acid) acid) 2 (amino 4 (amino acid)
acid)
[0369] Antibody 8.10.3F DNA sequence inserts were obtained from the
hybridoma cell line and subcloned into expression vectors. The
expression vectors were then transfected into a mouse myeloma (NS0)
host cell line to generate a primary transfectant cell line
producing anti-M-CSF antibodies having the heavy and light chain
sequences of 8.10.3F. Finally, samples of the 8.10.3F antibody
producing NS0 cell line were frozen and stored in liquid
nitrogen.
EXAMPLE 3
[0370] This Example shows the production of anti-M-CSF 8.10.3F
antibodies from the NS0 cell line generated according to Example
2.
[0371] A vial of 8.10.3F subcloned NS0 cells was removed from
liquid nitrogen storage as described in Example 2. The frozen cells
were thawed rapidly to 37.degree. C. until the last ice crystal
disappeared. The entire contents (1 milliliter) of the thawed vial
were then pipetted into a 75 cm.sup.2 T-Flask. Fourteen milliliters
of prewarmed (36.5.degree. C..+-.1.0.degree. C.) CD Hybridoma
growth medium (available from Invitrogen, Carlsbad, Calif.)
containing 10% Low IgG containing fetal bovine serum (available
from Invitrogen, Carlsbad, Calif.) was slowly pipetted into the
T-Flask.
[0372] The flask was planted at a target viable cell density of
from about 2.0.times.10.sup.5 to about 5.0.times.10.sup.5 cells/ml.
The flask was then placed in an incubator having a carbon dioxide
level of 9% and a temperature of 36.5.degree. C. and the cells were
grown for about 3 days. At the end of this period, targeted cell
number was on the order of 1.0 to 3.0.times.10.sup.6 cells/ml.
[0373] After the cells were grown for about 3 days, they were split
so that a target cell density of
2.5.times.10.sup.5+/-0.5.times.10.sup.5 was achieved and then
disposable shake flasks (i.e., seed flasks) were seeded based on
cell density. Each shake flask contained CD Hybridoma growth media
containing 10% Low IgG containing fetal bovine serum. The flasks
were then shaken at 100+/-10 rpm at 36.5.degree. C..+-.1.0.degree.
C. for about 3 days. Cell density in each flask at the end of this
period was 1.0 to 3.0.times.10.sup.6 cells/ml and greater than 80%
of the cells were viable.
[0374] After the cells were grown for about 3 days, the broth was
harvested. Clarified broth was obtained after centrifugation for 15
minutes at 7000 rpm and subsequent filtration with a sterile 0.22
.mu.m 4 inch Opticap.TM. Millipore.TM. filter into a sterile
TC-Tech.TM. bag.
EXAMPLE 4
[0375] This Example shows the purification of anti-M-CSF antibodies
from Example 3.
[0376] The clarified broth was then purified with three
chromatographic steps comprising a Protein A affinity column and
two ion exchange columns. A low pH inactivation and a viral
filtration were also done to clear any potential viruses in the
process. The product is concentrated and diafiltered into the
formulation buffer to make the anti-M-CSF antibody composition.
[0377] The Protein A column (Amersham Pharmacia) was prepped by
washing with 3 column volumes of 8 M urea, followed by an
equilibration wash with 20 mM Tris (pH 8). The final filtrate from
Example 3 was spiked with 2% v/v of 1M Tris pH 8.3 and 0.02%
NaN.sub.3 before being loaded onto the Protein A column via
gravity-drip mode. After load was complete, the resin was washed
with 5 column volumes of 20 mM Tris (pH 8), followed by 5 column
volumes of the elution buffer (0.1 M Glycine, pH 3.0). Any
precipitation was noted, and then a 10% v/v spike of 1M Tris pH 8.3
was added to the eluted antibody. The eluted protein was then
dialyzed into 100 fold the volume amount of eluted material of
dialysis buffer (e.g., 140 mM sodium chloride/20 mM sodium acetate,
pH 5.5). Following dialysis, the antibody was sterile filtered with
a 0.22 .mu.m filter and stored until further use.
EXAMPLE 5
[0378] A study was conducted to evaluate the effect of EDTA and
histidine on discoloration, aggregation, and fragmentation in
liquid compositions comprising monoclonal anti-M-CSF antibody
8.10.3F. Discoloration and aggregation in such liquid compositions
are generally undesirable from a product aesthetic perspective, a
product integrity perspective, or both.
Preparation of the Formulation
[0379] The pharmaceutical formulations of the invention were made
according to the following protocol. Materials which were used in
preparation of the formulations include: glacial acetic acid 99.9%
(Molecular Weight (MW) 60.05); concentrated sodium hydroxide 18.94N
(50% w/w; MW 40.0); concentrated hydrochloric acid 37.8% (12.44N;
MW 36.46); histidine (MW 155.16); sodium chloride (MW 58.44);
mannitol (MW 182.17); polysorbate 80 (Crillet.RTM. 4 HP); sodium
acetate trihydrate (MW 136.08); sodium citrate dihydrate (MW
294.1); disodium ethylenediaminetetraacetic acid dihydrate (MW
292.2); succinic acid (MW 118.1); antibody 8.10.3F bulk solution
(about 10 mg/ml in sodium acetate, pH 5.5, prepared according to
Examples 2-4); and water for injection (Milli-Q water). These
solutions were prepared and then sterile filtered into 1 L Nalgene
bottles and stored at 5.degree. C.
[0380] The antibody formulations that were evaluated are listed in
Table 3 below. To prepare each formulation, first the formulation
buffers were made either with buffer only or with buffer and
additional excipients such as tonicity agents (except addition of
surfactant such as polysorbate 80) (reported in Table 3), followed
by adjustment of pH to desired level. The buffer solutions were
then filtered through sterilizing filter (0.22 micron pore size)
into a sterilized receptacle. An antibody bulk solution from the
purification process described in Example 4 was obtained at about
10-15 mg/mL in 20 mM sodium acetate buffer pH 5.5 and 140 mM sodium
chloride. Buffer exchanges of this bulk solution into the above
identified formulation solutions were carried out with Amicon.RTM.
Centrifugal concentrators (e.g. with 30 kD cut-off membrane) on an
Eppendorf 5810R centrifuge run at about 4500.times.g. At least 8
volume exchanges were made for each formulation in respective
buffers. Approximately 2 to 5 milliliters of formulations 1 through
13 were prepared. Antibody concentrations were determined by
Ultraviolet-Visible spectrometry (UV-Vis) method using an
extinction coefficient of 1.43 (mg/ml).sup.-1 cm.sup.-1 at 280 nm.
The final volume of the antibody solution was adjusted by
appropriate dilution to achieve desired antibody concentration. A
20 mg/ml polysorbate 80 (PS80) solution was prepared by dilution
and dissolution of polysorbate 80 by the appropriate formulation
buffer prepared as described above. For formulations 9 to 13,
addition of required quantity of 20 g/L polysorbate 80 solution was
made to achieve 0.2 g/L polysorbate 80 in the antibody formulation.
The formulations with all of its ingredients included, was then
sterilized by filtration through sterile 0.22 micron membrane
filter.
[0381] For the formulation number 11 (i.e., histidine, mannitol,
polysorbate 80, and EDTA), a 1 molar (M) hydrochloric acid solution
was first prepared by appropriate dilution from concentrated
hydrochloric acid with water for injections. Individual solutions
were then prepared by dissolving the following pre-weighed
ingredients in about 90% of the water for injections: 45 grams per
liter (g/L) of mannitol, 1.55 g/L of histidine, 0.02 g/L of
disodium ethylenediaminetetraacetic acid dihydrate. After addition
of all of the excipients except polysorbate 80, dissolution was
achieved, and the pH of the solution was adjusted to pH 6 with 1M
hydrochloric acid solution which was prepared as described above.
After the addition of the hydrochloric acid solution, the final
quantity of the water was added. The buffer solution was then
filtered through a sterilization filter (0.22 micron pore size)
into a sterilized receptacle.
[0382] A 20 g/L polysorbate 80 solution was prepared by appropriate
dilution of polysorbate 80 by formulation buffer (45 g/L of
mannitol, 1.55 g/L of histidine, 0.02 g/L of disodium
ethylenediaminetetraacetic acid dihydrate, pH 6).
[0383] The filtered formulations were then filled into vials. The
vials were washed and autoclaved, as were the 13 mm Daikyo 777-1
serum stoppers. A fill-volume of 0.25 to 1 ml was used in 2 ml Type
1 glass vials. The vials were closed with Daikyo 777-1
Fluorotec.RTM. coated stoppers, crimp sealed, and placed in
stability chambers.
Formulation Appearance Analysis:
[0384] Each formulation was visually evaluated at initial (i.e.,
time zero) and thereafter at desired sampling intervals (weeks) for
particulate formation, color change, and turbidity change. Visual
observations were reported in Table 3. The appearance assays were
via visual inspection performed in a light box equipped with white
and black backgrounds. Antibody concentrations were determined by
ultraviolet-visible spectrometry (UV-Vis) methods using an
extinction coefficient of 1.34 (mg/ml)-1.cm-1 at 280 nm.
TABLE-US-00004 TABLE 3 Description of Anti-M-CSF 8.10.3F Antibody
Formulations, Appearance and Concentration. 8.10.3F Formu- Antibody
lation Formulation Visual concentration, No. Description pH
Evaluation (mg/ml) 1 20 mM sodium acetate 4.0 clear and 8.2
colorless 2 5 mM sodium acetate, 5 mM 5.0 clear and 11.9 sodium
citrate, 5 mM colorless histidine, 5 mM succinic acid 3 5 mM sodium
acetate, 5 mM 5.5 clear and 8.2 sodium citrate, 5 mM colorless
histidine, 5 mM succinic acid 4 20 mM histidine 6.0 clear and 8.2
colorless 5 20 mM sodium citrate 5.5 clear and 8.1 colorless 6 20
mM sodium acetate 5.5 clear and 8.1 colorless 7 20 mM sodium
succinate 5.5 clear and 8.4 colorless 8 20 mM disodium EDTA 5.5
clear and 8.1 dihydrate colorless 9 20 mM sodium acetate, 5.5 clear
and 8.4 140 mM NaCl, 0.2 mg/ml colorless polysorbate 80 10 10 mM
sodium acetate, 5.5 clear and 9.0 45 mg/ml Mannitol, colorless 0.02
mg/ml EDTA, 0.2 mg/ml polysorbate 80 11 10 mM histidine, 45 mg/ml
6.0 clear and 8.4 mannitol, 0.02 mg/ml colorless disodium EDTA
dihydrate, 0.2 mg/ml polysorbate 80 12 10 mM sodium citrate, 5.5
clear and 8.4 45 mg/ml mannitol, colorless 0.02 mg/ml disodium EDTA
dihydrate, 0.2 mg/ml polysorbate 80 13 10 mM sodium succinate, 5.5
clear and 8.6 45 mg/ml mannitol, colorless 0.02 mg/ml disodium EDTA
dihydrate, 0.2 mg/ml polysorbate 80
[0385] The results in Table 3 indicate that all tested antibody
8.3.1.degree. F. formulations had no significant discoloration, no
significant turbidity, and no significant particulate formation at
the initial timepoint (i.e., time equal zero).
EXAMPLE 6
[0386] A study was conducted to evaluate the effect of various
formulation compositions and pH on anti-M-CSF antibody 8.10.3F
fragmentation.
Fragmentation Analysis:
[0387] As noted above, the antibody formulations prepared according
to Table 3 and Example 5 were stored at a temperature of 40.degree.
C. At weeks, 0 (initial), 2, 4, and 6, the 40.degree. C.
formulations were analyzed for fragmentation using reduced SDS-PAGE
(rSDS-PAGE). The formulation vials were aseptically sampled at each
time point and an aliquot from the vial was loaded onto NuPAGE
4-12% bis-Tris gels with colloidal blue (Coomassie) stain. Gel
reduction was achieved by use of the NuPAGE.RTM. reducing agent.
Percentage fragmentation (i.e., the presence of an 11 kilodalton
(kD) polypeptide fragment and other fragments) in the reduced gels
was estimated densitometrically by 100% minus (% heavy chain+%
light chain) and reported in Table 4. FIG. 1 shows a line graph
that shows the percent fragmentation (i.e., presence of
polypeptides other than heavy chain (approx 50 kD) and light chain
(approx 25 kD)) estimated from SDS-PAGE reduced gels. Reduced
fragmentation was seen at pH ranges between 5.5 and 6.0. The gel
data showed fragment bands with approximate molecular masses of 40
kD and 11 kD.
TABLE-US-00005 TABLE 4 Percent Fragmentation for Formulations in
Table 3 after Storage at 40.degree. C.: Formulation 2 weeks 4 weeks
6 weeks No. Initial 40.degree. C. 40.degree. C. 40.degree. C. 1 0
7% 20.1% 24.5% 2 0 5.3% 13.1% 18.1% 3 0 2.6% 6.6% 10.8% 4 0 2.5%
10.9% 12% 5 0 2.4% 4.7% 2.2% 6 0 0 2.2% 2.6% 7 0 0 6.2% 5.5% 8 0
2.7% 8.8% 9.4% 9 0 -- -- 4.1% 10 0 -- -- 2.9% 11 0 -- -- 5.0% 12 0
-- -- 4.6% 13 0 -- -- 3.7%
[0388] FIG. 1 shows the percent fragmentation (i.e., presence of
polypeptides other than heavy chain (about 50 kD) and light chain
(about 25 kD) for each of the sample formulations detailed in Table
4. Reduced levels of fragmentation were seen in formulations having
pH ranges between 5.5 and 6.0. Reduced levels of fragmentation were
also seen in formulations without acetate, but having a chelating
agent.
EXAMPLE 7
[0389] A study was conducted to evaluate the effect of various
formulation compositions and pH on anti-M-CSF antibody 8.10.3F
charged species generation. Percentage major isoelectric focusing
(IEF) band estimated from IEF gels with antibody samples stored at
40.degree. C. over 6 weeks.
Formation of Acidic and Basic Species:
[0390] Antibody formulations 1-4 prepared according to Table 3 and
Example 5 were stored at a temperature of 40.degree. C. After
storing for 6 weeks, each formulation was analyzed for the
formation of acidic and basic species using Isoelectric Focusing
(IEF). The Imaging Capillary Electrophoresis was conducted using a
Convergent Biosciences iCE.sub.280 analyzer for evaluation of
charge heterogeneity. The Convergent iCE.sub.280 is an imaging
capillary isoelectric focusing (IEF) instrument, which allows the
user to take an image of a separated sample contained within a
capillary. IEF assays were conducted using pH 3-10.5 polyacrylamide
gels and Coomassie blue stain. The sample protein components were
separated based on their relative isoelectric points (pI). The
major species was assigned based on the highest densitometric band
intensity at a particular pI, in the initial samples. The change in
percentage major species was followed as a function of storage
duration. The loss in percentage major species from the initial
value is a measure of the extent of acidic and basic specied
formation.
[0391] Formation of acidic and basic species was also monitored by
Imaging Capillary Electrophoresis (iCE). ICE was conducted using a
Convergent Biosciences iCE.sub.280 analyzer for evaluation of
charge heterogeneity. The Convergent iCE.sub.280 is an imaging
capillary isoelectric focusing instrument, which allows the user to
take an image of a separated sample contained within a capillary.
The samples were prepared in a mixture of electrophoretic
ampholytes, methyl cellulose, calibration markers, and water. The
samples were introduced into the iCE.sub.280 and a high
potential/voltage was applied. The sample protein components were
separated based on their relative isoelectric points (pI). The
relative amount of each separated component was observed by an
imaging CCD camera. The data was then processed and reported as
loss of the main peak (i.e., formation of acidic and basic species)
using conventional chromatography integration software.
[0392] FIG. 2 shows the percentage major IEF band estimated from
IEF gels with formulations 1-4 stored at 40.degree. C. over 6
weeks. As seen in FIG. 2, a lesser extent of decrease in the major
IEF band at pH 5.5 and 6.0 suggested improved stability at pH
ranging from 5.5 and 6.0 (i.e., formulation nos. 3 and 4).
EXAMPLE 8
[0393] A study was conducted to evaluate the effect of EDTA on
anti-M-CSF antibody 8.10.3F aggregation.
[0394] Specifically, sample formulation nos. 3, 5, 6, 7, and 8 were
prepared with and without EDTA according to Table 3 and Example 5
and stored in several glass vials at 40.degree. C. for 0 (initial),
2, 4, and 6 weeks. The glass vials were then sampled aseptically to
measure the level of antibody 8.11.3F aggregation in the
formulations at the 0, 2, 4, and 6 week time points. In addition,
formulation 11 (with EDTA) was also prepared according to Table 3
and stored in several glass vials at 40.degree. C. for 26
weeks.
Aggregation Analysis:
[0395] After weeks 0, 2, 4, and 6, each formulation was analyzed
for aggregation using size exclusion chromatography. The size
exclusion chromatography (i.e., SE-HPLC) was carried out using a
TSK gel G3000SWXL-G2000SWXL column, mobile phase 0.2 M sodium
phosphate buffer at pH 7.0, a flow rate of 1 ml/min, and UV
detection at 214 nm. Table 5 shows the percentage of eluted high
molecular weight species (i.e., aggregates of anti-M-CSF antibody
8.11.3F) measured at the relevant times for each of the formulation
treatments. Aggregation levels were calculated by integrating the
areas under the chromatogram peaks for each formulation and
reporting the integrated areas under the high molecular weight
species peaks as a percentage of total peak area (see Table 5).
TABLE-US-00006 TABLE 5 Percent Aggregation for Formulations 3, 5,
6, 7, and 8 after Storage at 40.degree. C.: Formulation 2 weeks 4
weeks 6 weeks No. Initial 40.degree. C. 40.degree. C. 40.degree. C.
3 0.2% 1.3% 1.9% 3.2% 5 0.2% 1.5% 2.2% 3.8% 6 0.2% 1.1% 1.9% 3% 7
0.2% 1.2% -- 2.9% 8 0.2% 1.1% 1.4% 2.6%
[0396] As can be seen in Table 5 and FIG. 3, the EDTA containing
formulation (i.e., formulation 8) showed lower levels of
aggregation over time as compared to formulations without EDTA.
FIG. 11 shows a size exclusion chromatogram for monoclonal
anti-M-CSF antibody 8.10.3F stored in formulation 11 for 26 weeks
at 40.degree. C.
EXAMPLE 9
[0397] A study was conducted to evaluate the effect of EDTA on
anti-M-CSF antibody 8.10.3F aggregation and fragmentation.
[0398] Specifically, sample formulation nos. 9, 10, 11, 12, and 13
were prepared with and without EDTA according to Table 3 and
Example 5 and stored in several glass vials at 40.degree. C. for 0
(initial), 4, 6, 8, 12 and 26 weeks. The glass vials were then
sampled aseptically to measure the level of antibody 8.11.3F
aggregation in the formulations at the predetermined time
points.
Aggregation Analysis:
[0399] At weeks 0, 4, 6, 8, 12, and 26, each formulation was
analyzed for aggregation using size exclusion chromatography. Size
exclusion--high pressure liquid chromatography (SE-HPLC) was
carried out using a TSK gel G3000SWXL-G2000SWXL column, mobile
phase 0.2 M sodium phosphate buffer at pH 7.0, a flow rate of 1
ml/min, and UV detection at 214 nm. Table 6 shows the percentage of
eluted high molecular weight species (i.e., aggregates of
anti-M-CSF antibody 8.11.3F) measured at the relevant times for
each of the formulation treatments. Aggregation levels were
calculated by integrating the areas under the chromatogram peaks
for each formulation and reporting the integrated areas under the
high molecular weight species peaks as a percentage of total peak
area (see Table 6).
TABLE-US-00007 TABLE 6 Percent Aggregation for Formulations 9, 10,
11, 12, and 13 after Storage at 40.degree. C.: Formulation 4 weeks
6 weeks 8 weeks 12 weeks 26 weeks No. Initial 40.degree. C.
40.degree. C. 40.degree. C. 40.degree. C. 40.degree. C. 9 0.2% 2.0%
2.6% 3.9% 5.8% 11.6% 10 0.1% 0.9% 1.4% 1.6% 2.7% 4.7% 11 0.2% 1.0%
1.4% 1.8% 2.6% 4.9% 12 0.2% 1.8% 2.3% 2.9% 3.9% 7.0% 13 0.2% 1.4%
1.9% 2.4% 3.5% 6.2%
[0400] As can be seen in Table 6 and FIG. 4, the EDTA containing
formulations (i.e., formulations 10, 11, 12 and 13) showed lower
levels of aggregation over time as compared to the formulation
without EDTA (i.e., formulation 9).
Fragmentation Analysis:
[0401] At weeks 0, 4, 6, 8, 12, and 26, formulation nos. 9, 10, 11,
12, and 13 were also analyzed for fragmentation.
[0402] Organic size exclusion--high pressure liquid chromatography
(SE-HPLC) was conducted on the samples at time points 0, 4, 6, 8,
12 and 26 weeks to determine the percent fragmentation for an
approximately 11 kD fragment of the antibody. The samples were
injected onto a TSK gel Super SW3000 size exclusion column, using
an isocratic mobile phase of 40% acetonitrile+0.1% TFA at a flow
rate of 0.50 mL/min and UV detection at 214 nm. The percentage of
eluted species was determined by integrating area under peaks.
TABLE-US-00008 TABLE 7 Percent Fragmentation of a between 10.5 kD
and a 11. kD Fragment for Formulations 9, 10, 11, 12, and 13 after
Storage at 40.degree. C.: Formulation 8 weeks 12 weeks 26 weeks No.
Initial 40.degree. C. 40.degree. C. 40.degree. C. 9 0.1% 1.6% 2.3%
3.5% 10 0.1% 0.7% 0.9% 1.7% 11 0.1% 0.6% 0.8% 1.7% 12 0.1% 1.5%
2.1% 4.1% 13 0.1% 1.0% 1.4% 3.0%
[0403] As can be seen in Table 7 and FIG. 5, the EDTA containing
formulations (i.e., formulations 10, 11, 12 and 13) showed lower
levels of fragmentation yielding an 11 kD fragment over time as
compared to the formulation without EDTA (i.e., formulation 9). In
addition, the histidine containing formulation (i.e., formulation
11) showed lower levels of fragmentation yielding an 11 kD fragment
over time as compared to the formulations without histidine (i.e.,
formulations 9, 12 and 13).
[0404] In addition, SDS-PAGE gels were also run with the samples at
time points 0, 4, 6, 8, 12 and 26 weeks using NuPAGE 4-12% Bis-Tris
gel, and colloidal blue (Coomassie) stain. For the reduced gels,
reduction was achieved by NuPAGE.RTM. reducing agent. Percentage
fragmentation in reduced gels was estimated densitometrically by
100% minus (% heavy chain+% light chain). The gel data showed
fragment bands with approximate molecular masses of 40 kD and 11
kD. FIG. 6 shows the percentage fragmentation estimated from
SDS-PAGE reduced gel data with the formulation samples. FIG. 7
shows the percentage monomer of antibody estimated from SDS-PAGE
non-reduced gel data with the formulation samples.
[0405] FIGS. 4-7 show improved anti-M-CSF antibody stability for
formulation 11 (10 mM histidine, 45 mg/ml mannitol, 0.02 mg/ml
disodium EDTA dihydrate, and 0.2 mg/ml polysorbate 80) for reduced
aggregation (FIG. 4), reduced quantity of 11 kD fragmentation (FIG.
5), reduced fragmented species (FIG. 6), and retaining highest %
intact antibody monomer (FIG. 7) as compared to formulations 9, 10,
12 and 13.
EXAMPLE 10
[0406] A study was conducted to evaluate the effect of EDTA and
histidine on anti-M-CSF antibody 8.10.3F fragmentation.
[0407] Several experimental formulations of antibody 8.10.3F
generated truncated species (i.e., fragments) upon stressed
conditions of 40.degree. C. for 6 weeks, as observed by the
formation of bands at approximately 40 kD and 11 kD appearing on
reduced SDS-PAGE gel photograph as shown in FIG. 9. The identity of
the most abundant clip site was determined to be between residues
Asp99 and Pro100 on the heavy chain of the molecule. Also observed
were some minor truncation sites in the light chain, one between
residues Gly213 and Glu214 and another between Glu214 and Cys215.
The truncation level varies depending on the formulation and thus
far has only been observed at higher temperatures (e.g., 40.degree.
C.) i.e., under stressed conditions.
[0408] A sample of antibody 8.10.3F, which was formulated in sodium
acetate and sodium chloride (formulation no. 9) and stored at
40.degree. C. for 26 weeks, was observed to have a higher presence
of an 11 kD fragment than a formulation comprising histidine and
EDTA (formulation no. 11) using organic SE-HPLC (see FIG. 8).
[0409] This sample was then analyzed by organic size exclusion
chromatography/mass spectrometry (SEC/MS) in order to determine the
site of truncation. The sample was injected onto a size exclusion
column (Phenomenex SEC3000, 4.6.times.250 mm) using an isocratic
mobile phase of 40% acetonitrile+0.1% TFA at a flow rate of 0.50
mL/min. The eluent of the column was split such that approximately
half of the flow was directed into the source of an electrospray
mass spectrometer (Micromass Q-T of Micro.TM., Waters Inc.). Mass
spectra of each of the chromatographic peaks were deconvoluted
using the MaxEnt algorithm included in the operating software. The
measured molecular masses were then compared to the theoretical
molecular mass based on the predicted amino acid sequence of
antibody 8.10.3F.
[0410] Organic SE-HPLC separation with 214 nm detection followed by
mass spectrometric identification were performed for antibody
8.10.3F in formulation no. 1 stored at 40.degree. C. for 6 weeks
compared to a control sample. FIG. 10 shows the resulting
chromatogram having the 40.degree. C. storage as the top graph and
the 5.degree. C. control as the bottom graph. The chromatogram
measured masses were tabulated and compared to the theoretical
masses of the postulated species in Table 8.
TABLE-US-00009 TABLE 8 The measured masses of the species observed
in the sample compared with the theoretical masses of the
postulated species. All of the measured molecular weights were
within experimental error of the theoretical molecular weights of
the postulated species. Peak RT (retention time) Measured
Theoretical (minutes) MW (Da) Identity MW (Da) 4.45 147,220 8.10.3F
147,223* ("Parent") 4.45 136,424 Parent minus 136,431* (Heavy Chain
1-99) 4.45 125,634 Parent minus 125,633* clipped 2x (Heavy Chain
1-99) 6.53 10,816 Heavy chain 1-99 10,816 *Consistent with "G0, G0"
glycoform and des-Lys C-terminus on heavy chains. The "G0, G1";
"G1, G1"; and "G1, G2" glycoforms were also observed. The "G0, G0"
glycoform is a species in which both heavy chains have the G0
glycan attached, as described in Jefferis et al., Biochem. J., 268,
529-537, (1990). The G1 and G2 glycans have one and two,
respectively, galactose residues on the nonreducing end of the
glycan. The antibodies were N-glycosylated at residue 297 of the
heavy chain.
[0411] Under stressed conditions, antibody 8.10.3F can undergo
cleavage and generate truncated species. The main cleavage site is
consistent with cleavage of an an Asp-Pro bond in the heavy chain
of 8.10.3F, which would generate a 10,816 Da (i.e., about 11 kD)
species along with the corresponding parent species minus one and
two of the truncation product.
EXAMPLE 11
[0412] A study was conducted to evaluate the effect of various
buffers on anti-M-CSF antibody 8.10.3F aggregation.
[0413] Specifically, sample formulation nos. 6, 3, 5 and 8 were
prepared with according to Table 9 and Example 5 and stored in
glass vials at 40.degree. C. for 6 weeks. The glass vials were then
sampled aseptically to measure the level of antibody 8.11.3F
aggregation in the formulations at the 6 week time point.
Aggregation Analysis:
[0414] At the 6 week time point, each formulation was analyzed for
aggregation using size exclusion chromatography. The size exclusion
chromatography (i.e., SE-HPLC) was carried out using a TSK gel
G3000SWXL-G2000SWXL column, mobile phase 0.2 M sodium phosphate
buffer at pH 7.0, a flow rate of 1 ml/min, and UV detection at 214
nm. Table 9 shows the percentage of eluted high molecular weight
species (i.e., aggregates of anti-M-CSF antibody 8.11.3F) measured
at the relevant time for each of the formulation treatments.
Aggregation levels were calculated by integrating the areas under
the chromatogram peaks for each formulation and reporting the
integrated areas under the high molecular weight species peaks that
eluted prior to the antibody monomer (i.e., the intact unaggregated
polypeptide) as a percentage of total peak area (see Table 9).
TABLE-US-00010 TABLE 9 Percent Aggregation for Formulations 6, 3, 5
and 8 after Storage at 40.degree. C.: 8.10.3F Antibody Formulation
concentration, Formulation No. (mg/ml) Description % Aggregation 6
8 .+-. 2 20 mM acetate pH 5.5 3.0% 3 8 .+-. 2 Combination, pH 5.5
3.2% (acetate 5 mM, citrate 5 mM, histidine 5 mM, succinate 5 mM) 5
8 .+-. 2 20 mM citrate, pH 5.5 3.8% 8 8 .+-. 2 20 mM EDTA, pH 5.5
2.6%
[0415] As can be seen in Table 9, the EDTA containing formulation
(i.e., formulation 8) showed reduced levels of aggregation as
compared to the formulations without EDTA (i.e., formulations 6, 3
and 5).
EXAMPLE 12
[0416] A study was conducted to evaluate the effect of various
excipients on anti-M-CSF antibody 8.10.3F aggregation and
fragmentation.
[0417] Specifically, sample formulation nos. 18, 19, 20, 29, 30 and
31 were prepared according to Table 10 and Example 5 and stored in
glass vials at 40.degree. C. for 6 weeks. The glass vials were then
sampled aseptically to measure the level of antibody 8.11.3F
aggregation and fragmentation in the formulations at the 6 week
time point.
Aggregation Analysis:
[0418] At the 6 week time point, each formulation was analyzed for
aggregation using size exclusion chromatography. The size exclusion
chromatography (i.e., SE-HPLC) was carried out using a TSK gel
G3000SWXL-G2000SWXL column, mobile phase 0.2 M sodium phosphate
buffer at pH 7.0, a flow rate of 1 ml/min, and UV detection at 214
nm. Table 10 shows the percentage of eluted high molecular weight
species (i.e., aggregates of anti-M-CSF antibody 8.11.3F) measured
at the relevant time for each of the formulation treatments.
Aggregation levels were calculated by integrating the areas under
the chromatogram peaks for each formulation and reporting the
integrated areas under the high molecular weight species peaks that
eluted prior to the antibody monomer (i.e., the intact unaggregated
polypeptide) as a percentage of total peak area (see Table 10).
Fragmentation Analysis:
[0419] At the 6 week time point, each formulation was also analyzed
for fragmentation using organic SE-HPLC. Organic SE-HPLC was
conducted on the samples to determine the percent fragmentation for
an 11 kD fragment of the total polypeptide. The samples were
injected onto a TSK gel Super SW3000 size exclusion column, using
an isocratic mobile phase of 40% acetonitrile+0.1% TFA at a flow
rate of 0.50 mL/min and UV detection at 214 nm. The percentage of
eluted species was determined by integrating area under peaks and
reported in Table 10.
TABLE-US-00011 TABLE 10 Description of Anti-M-CSF 8.10.3F Antibody
Formulations and Results after Storage at 40.degree. C.:
Formulation 8.10.3F % % No. mg/ml Buffer Excipient PS80
Na.sub.2EDTA.cndot.2H.sub.2O Aggregate Fragment 29 6 .+-. 2 Sodium
Sodium 0.2 -- 2.0% 1.2% acetate, chloride 20 mM, 140 mM pH 5.5 18 6
.+-. 2 Sodium Mannitol 0.2 -- 1.4% 1.0% acetate, 45 mg/ml 20 mM, pH
5.5 30 6 .+-. 2 Sodium Mannitol 0.2 0.02 1.3% 0.6% acetate, 45
mg/ml mg/ml 20 mM, pH 5.5 19 6 .+-. 2 Sodium Sucrose 0.2 0.02 1.2%
0.5% acetate, 90 mg/mL mg/ml 20 mM, pH 5.5 20 6 .+-. 2 Sodium
Trehalose 0.2 0.02 1.1% 0.4% acetate, 90 mg/mL mg/ml 20 mM, pH 5.5
31 6 .+-. 2 Histidine, Mannitol 0.2 0.02 0.8% 0.5% 10 mM, 45 mg/ml
mg/ml pH 6.0
[0420] As can be seen in Table 10, the EDTA containing formulations
(i.e., formulations 30, 31, 19, and 20) showed reduced levels of
aggregation and fragmentation as compared to the formulations
without EDTA (i.e., formulations 29 and 18).
EXAMPLE 13
[0421] A study was conducted to evaluate the effect of various
excipients on anti-M-CSF antibody 8.10.3F fragmentation.
[0422] Specifically, sample formulation nos. 21-28 were prepared
according to Table 11 and Example 5 and stored in glass vials at
40.degree. C. for 26 weeks. The glass vials were then sampled
aseptically to measure the level of antibody 8.11.3F fragmentation
in the formulations at the 26 week time point.
Fragmentation Analysis:
[0423] At the 26 week time point, each formulation was also
analyzed for fragmentation using organic SE-HPLC. Organic SE-HPLC
was conducted on the samples to determine the percent fragmentation
for an 11 kD fragment of the total polypeptide. The samples were
injected onto a TSK gel Super SW3000 size exclusion column, using
an socratic mobile phase of 40% acetonitrile+0.1% TFA at a flow
rate of 0.50 mL/min and UV detection at 214 nm. The percentage of
eluted species was determined by integrating area under peaks and
reported in Table 11.
TABLE-US-00012 TABLE 11 Description of Anti-M-CSF 8.10.3F Antibody
Formulations and Results after Storage at 40.degree. C.:
Formulation 8.10.3F PS80 Na.sub.2EDTA.cndot.2H.sub.2O % No. mg/ml
Buffer Excipients mg/mL mg/mL Fraqmentation 21 50 .+-. 8 Sodium
Sodium 0.2 -- 4.1% acetate, chloride 20 mM, 140 mM pH 5.5 22 50
.+-. 8 Sodium Mannitol 0.2 0.02 2.3% acetate, 45 mg/ml mg/ml 10 mM,
pH 5.5 23 50 .+-. 8 Sodium Sucrose 0.2 0.02 3.0% acetate, 90 mg/ml
mg/ml 10 mM, pH 5.5 24 50 .+-. 8 Sodium Trehalose 0.2 0.02 2.2%
acetate, 90 mg/ml mg/ml 10 mM, pH 5.5 25 50 .+-. 8 Histidine,
Mannitol 0.2 0.02 2.1% 10 mM, 45 mg/ml mg/ml pH 6.0 26 50 .+-. 8
Histidine, Sucrose 0.2 0.02 1.9% 10 mM, 90 mg/ml mg/ml pH 6.0 27 50
.+-. 8 Histidine, Trehalose 0.2 0.02 1.8% 10 mM, 90 mg/ml mg/ml pH
6.0 28 50 .+-. 8 Histidine, Mannitol 0.2 0.02 1.4% 5 mM, 45 mg/ml
mg/ml pH 6.0
[0424] As can be seen in Table 11, the EDTA containing formulations
(i.e., formulations 22-28) showed reduced levels of fragmentation
as compared to the formulation without EDTA (i.e., formulation 21).
Likewise, as can be seen in Table 11, the histidine containing
formulations (i.e., formulations 25-28) showed reduced levels of
fragmentation as compared to the formulations without histidine
(i.e., formulations 21-24).
[0425] All references cited in this specification, including
without limitation all papers, publications, patents, patent
applications, presentations, texts, reports, manuscripts,
brochures, books, internet postings, journal articles, periodicals,
and the like, are hereby incorporated by reference into this
specification in their entireties. The discussion of the references
herein is intended merely to summarize the assertions made by their
authors and no admission is made that any reference constitutes
prior art. Applicants reserve the right to challenge the accuracy
and pertinency of the cited references.
[0426] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description
shall be interpreted as illustrative and not in a limiting sense.
In addition, it should be understood that aspects of the various
embodiments may be interchanged both in whole or in part.
SEQUENCES
TABLE-US-00013 [0427] SEQ ID NO: 1
atggagttggggctgtgctgggttttccttgttgctattttagaaggtgt
ccagtgtgaggtgcagctggtggagtctgggggaggcttggtacagcctg
gggggtccctgagactctcctgtgcagcctctggattcaccttcagtagt
tttagtatgacctgggtccgccaggctccaggaaaggggctggagtgggt
ttcatacattagtagtagaagtagtaccatatcctacgcagactctgtga
agggccgattcaccatctccagagacaatgccaagaactcactgtatctg
caaatgaacagcctgagagacgaggacacggctgtgtattactgtgcgag
agatcctcttctagcgggagctaccttctttgactactggggccagggaa
ccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccc
ctggcgccctgctccaggagcacctccgagagcacagcggccctgggctg
cctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcag
gcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctca
ggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcgg
cacccagacctacacctgcaacgtagatcacaagcccagcaacaccaagg
tggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgccca
gcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtgg
acgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggc
gtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacag
cacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctga
acggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagccccc
atcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggt
gtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcc
tgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgg
gagagcaatgggcagccggagaacaactacaagaccacacctcccatgct
ggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga
gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct
ctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ ID NO: 2
MELGLCWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSS
FSMTWVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYL
QMNSLRDEDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSASTKGPSVFP
LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP
IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
SEQ ID NO: 3 atggaaaccccagcgcagcttctcttcctcctgctactctggctcccaga
taccaccggagaatttgtgttgacgcagtctccaggcaccctgtctttgt
ctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagc
agcagttacttagcctggtaccagcagaaacctggccaggctcccaggct
cctcatctatggtgcatccagcagggccactggcatcccagacaggttca
gtggcagtgggtctgggacagacttcactctcaccatcagcagactggag
cctgaagattttgcagtgtattactgtcagcagtatggtagctcacctct
cactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcac
catctgtcttcatcttcccgccatctgatgagcagttaaatctggaactg
cctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagta
cagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt
cacagagcaggacagcaaggacagcacctacagcctcagcagcaccctga
cgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtc
acccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggaga gtgt SEQ ID NO:
4 METPAQLLFLLLLWLPDTTGEFVLTQSPGTLSLSPGERATLSCRASQSVS
SSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Sequence CWU 1
1
411398DNAHomo sapiens 1atggagttgg ggctgtgctg ggttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcagctgg tggagtctgg gggaggcttg gtacagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggattcac cttcagtagt
tttagtatga cctgggtccg ccaggctcca 180ggaaaggggc tggagtgggt
ttcatacatt agtagtagaa gtagtaccat atcctacgca 240gactctgtga
agggccgatt caccatctcc agagacaatg ccaagaactc actgtatctg
300caaatgaaca gcctgagaga cgaggacacg gctgtgtatt actgtgcgag
agatcctctt 360ctagcgggag ctaccttctt tgactactgg ggccagggaa
ccctggtcac cgtctcctca 420gcctccacca agggcccatc ggtcttcccc
ctggcgccct gctccaggag cacctccgag 480agcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540tggaactcag
gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca
600ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg
cacccagacc 660tacacctgca acgtagatca caagcccagc aacaccaagg
tggacaagac agttgagcgc 720aaatgttgtg tcgagtgccc accgtgccca
gcaccacctg tggcaggacc gtcagtcttc 780ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacgtgc 840gtggtggtgg
acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc
900gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag
cacgttccgt 960gtggtcagcg tcctcaccgt tgtgcaccag gactggctga
acggcaagga gtacaagtgc 1020aaggtctcca acaaaggcct cccagccccc
atcgagaaaa ccatctccaa aaccaaaggg 1080cagccccgag aaccacaggt
gtacaccctg cccccatccc gggaggagat gaccaagaac 1140caggtcagcc
tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg
1200gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct
ggactccgac 1260ggctccttct tcctctacag caagctcacc gtggacaaga
gcaggtggca gcaggggaac 1320gtcttctcat gctccgtgat gcatgaggct
ctgcacaacc actacacgca gaagagcctc 1380tccctgtctc cgggtaaa
13982466PRTHomo sapiens 2Met Glu Leu Gly Leu Cys Trp Val Phe Leu
Val Ala Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser Phe Ser Met Thr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ser Tyr Ile
Ser Ser Arg Ser Ser Thr Ile Ser Tyr Ala65 70 75 80Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val 100 105 110Tyr
Tyr Cys Ala Arg Asp Pro Leu Leu Ala Gly Ala Thr Phe Phe Asp 115 120
125Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu145 150 155 160Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn 210 215 220Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg225 230 235
240Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
245 250 255Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile 260 265 270Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu 275 280 285Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His 290 295 300Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe Arg305 310 315 320Val Val Ser Val Leu
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys 325 330 335Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu 340 345 350Lys
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360
365Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
370 375 380Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp385 390 395 400Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met 405 410 415Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 420 425 430Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 435 440 445Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460Gly
Lys4653705DNAHomo sapiens 3atggaaaccc cagcgcagct tctcttcctc
ctgctactct ggctcccaga taccaccgga 60gaatttgtgt tgacgcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 120ctctcctgca gggccagtca
gagtgttagc agcagttact tagcctggta ccagcagaaa 180cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
240gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 300cctgaagatt ttgcagtgta ttactgtcag cagtatggta
gctcacctct cactttcggc 360ggagggacca aggtggagat caaacgaact
gtggctgcac catctgtctt catcttcccg 420ccatctgatg agcagttgaa
atctggaact gcctctgttg tgtgcctgct gaataacttc 480tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc
540caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag
cagcaccctg 600acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 660ggcctgagct cgcccgtcac aaagagcttc
aacaggggag agtgt 7054235PRTHomo sapiens 4Met Glu Thr Pro Ala Gln
Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu
Phe Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Ser Ser
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg
Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro65 70 75
80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr 100 105 110Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 115 120 125Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe145 150 155 160Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200
205Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
235
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