U.S. patent application number 10/222140 was filed with the patent office on 2004-02-19 for formulation of human antibodies for treating tnf-alpha associated disorders.
Invention is credited to Baust, Lisa, Dickes, Michael, Krause, Hans-Juergen.
Application Number | 20040033228 10/222140 |
Document ID | / |
Family ID | 31714885 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033228 |
Kind Code |
A1 |
Krause, Hans-Juergen ; et
al. |
February 19, 2004 |
Formulation of human antibodies for treating TNF-alpha associated
disorders
Abstract
A liquid aqueous pharmaceutical formulation is described which
has a high protein concentration, a pH of between about 4 and about
8, and enhanced stability.
Inventors: |
Krause, Hans-Juergen;
(Worms, DE) ; Baust, Lisa; (Mannheim, DE) ;
Dickes, Michael; (Rodersheim-Gronau, DE) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
31714885 |
Appl. No.: |
10/222140 |
Filed: |
August 16, 2002 |
Current U.S.
Class: |
424/145.1 |
Current CPC
Class: |
A61K 39/39591 20130101;
A61P 9/00 20180101; A61P 9/04 20180101; A61P 35/00 20180101; A61P
19/06 20180101; A61K 47/12 20130101; A61K 47/22 20130101; A61P 1/04
20180101; A61P 1/02 20180101; A61P 9/10 20180101; A61P 31/00
20180101; A61P 11/06 20180101; A61P 31/04 20180101; A61K 9/19
20130101; A61P 1/00 20180101; A61P 1/18 20180101; C07K 2317/76
20130101; A61K 47/02 20130101; A61K 9/0019 20130101; A61P 11/00
20180101; A61P 27/02 20180101; A61K 2039/505 20130101; A61K 47/26
20130101; A61P 37/08 20180101; C07K 2317/21 20130101; A61P 25/02
20180101; Y02A 50/30 20180101; A61K 39/3955 20130101; A61P 31/18
20180101; A61P 29/00 20180101; A61P 13/12 20180101; A61P 7/00
20180101; A61P 37/06 20180101; A61P 43/00 20180101; A61P 3/10
20180101; A61P 1/16 20180101; A61P 33/06 20180101; A61P 19/08
20180101; A61P 3/04 20180101; C07K 16/241 20130101; C07K 2317/92
20130101; A61P 19/04 20180101; A61K 47/10 20130101; A61P 17/00
20180101; A61P 19/02 20180101; A61P 15/00 20180101; A61P 17/06
20180101; A61P 31/12 20180101; A61P 37/02 20180101; C07K 2317/94
20130101; A61P 37/00 20180101; A61P 25/04 20180101 |
Class at
Publication: |
424/145.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A pharmaceutical formulation selected frrom the group consisting
of: (a) A liquid aqueous pharmaceutical formulation comprising a
therapeutically effective amount of an antibody in a buffered
solution, said formulation having a pH between about 4 and 8 and
having a shelf life of at least 18 months. (b) An aqueous
pharmaceutical formulation comprising a therapeutically effective
amount of an antibody in a buffered solution, said formulation
having a pH between about 4 and 8 and having a shelf life of at
least 18 months in the liquid state. (c) A liquid aqueous
pharmaceutical formulation comprising a therapeutically effective
amount of an antibody in a buffered solution, said formulation
having a pH between about 4 and 8 which maintains stability
following at least 3 freeze/thaw cycles of the formulation. (d) A
liquid aqueous pharmaceutical formulation comprising a
therapeutically effective amount of an antibody in a buffered
solution, said formulation having a pH between 4 and 8 and having
enhanced stability of at least 12 months at a temperature of
2-8.degree. C.
2. The formulation of claim 1, wherein the antibody is directed to
TNF.alpha..
3. The formulation of claim 1, wherein the concentration of the
antibody is between about 1-150 mg/ml.
4. The formulation of claim 1, wherein the concentration of the
antibody is about 50 mg/ml.
5. The formulation of claim 1, which further is suitable for single
use sc injection.
6. The formulation of claim 1, wherein the antibody is an antibody,
or an antigen-binding portion thereof, that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less.
7. The formulation of claim 6, wherein the antibody, or
antigen-binding portion thereof, is a recombinant antibody, or
antigen-binding portion thereof.
8. The formulation of claim 1, wherein the antibody is an the
antibody, or antigen-binding portion, thereof which: a) dissociates
from human TNF.alpha. with a K.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, as determined by surface
plasmon resonance; b) has a light chain CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3
by a single alanine substitution at position 1, 4, 5, 7 or 8 or by
one to five conservative amino acid substitutions at positions 1,
3, 4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domain comprising
the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID
NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6,
8, 9, 10 or 11 or by one to five conservative amino acid
substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or
12.
9. The formulation of claim 1, wherein the antibody, or
antigen-binding portion thereof, has a light chain variable region
(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a
heavy chain variable region (HCVR) comprising the amino acid
sequence of SEQ ID NO: 2.
10. The formulation of claim 1, wherein the antibody, or
antigen-binding portion thereof, neutralizes the activity of human
TNF.alpha., chimpanzee TNF.alpha. and at least one additional
primate TNF.alpha. selected from the group consisting of baboon
TNF.alpha., marmoset TNF.alpha., cynomolgus TNF.alpha. and rhesus
TNF.alpha..
11. The formulation of claim 1, wherein the antibody, or an
antigen-binding portion thereof, also neutralizes the activity of
mouse TNF.alpha. and/or pig TNF.alpha..
12. The formulation of claim 1, wherein the antibody, or
antigen-binding portion thereof, binds human TNF.alpha. and is the
antibody D2E7 or an antigen binding portion thereof.
13. An aqueous pharmaceutical composition comprising a polyol, a
surfactant, and a buffer system comprising citrate and/or phosphate
with a pH of about 4 to 8, in amounts sufficient to formulate an
antibody for therapeutic use at a concentration of greater than
about 45 mg/ml.
14. The composition of claim 13, wherein the polyol is mannitol and
the surfactant is polysorbate 80.
15. The composition of claim 14, which contains 5-20 mg/ml of
mannitol and 0.1-10 mg/ml of polysorbate 80.
16. The formulation of claim 13, which contains an antibody, or
antigen-binding portion thereof, which binds human TNF.alpha. and
is the antibody D2E7 or an antigen binding portion thereof.
17. A liquid aqueous pharmaceutical formulation comprising (a)
1-150 mg/ml of antibody, (b) 5-20 mg/ml of mannitol, (c) 0.1-10
mg/ml of Tween-80, and (d) a buffer system comprising citrate
and/or phosphate, with a pH of 4 to 8.
18. The formulation of claim 17, wherein the pH is selected from
the group consisting of between about 4.5 to about 6.0, between
about 4.8 to about 5.5, and between about 5.0 to about 5.2.
19. The liquid aqueous pharmaceutical formulation of claim 17,
which contains (a) about 50 mg/ml of antibody, (b) about 12 mg/ml
of mannitol, (c) about 1 mg/ml of Tween-80, and
20. The formulation of claim 17, wherein the buffer system
comprises (a) about 1.3 mg/ml of citric acid, (b) about 0.3 mg/ml
of sodium citrate, (c) about 1.5 mg/ml of disodium phosphate
dihydrate, (d) about 0.9 mg/ml of sodium dihydrogen phosphate
dihydrate, and (e) about 6.2 mg/ml of sodium chloride
21. The formulation of claim 19, wherein the antibody is directed
to TNF.alpha..
22. The formulation of claim 19, wherein the antibody, or
antigen-binding portion thereof, binds human TNF.alpha. and is the
antibody D2E7 or an antigen binding portion thereof.
23. The formulation of claim 22, which is administered to a subject
suffering from a disorder in which TNF.alpha. activity is
detrimental such that TNF.alpha. activity in the subject is
inhibited
Description
BACKGROUND OF THE INVENTION
[0001] Tumor necrosis factor .alpha. (TNF.alpha.) is a cytokine
produced by numerous cell types, including monocytes and
macrophages, that was originally identified based on its capacity
to induce the necrosis of certain mouse tumors (see e.g., Old, L.
(1985) Science 230:630-632). Subsequently, a factor termed
cachectin, associated with cachexia, was shown to be the same
molecule as TNF.alpha.. TNF.alpha. has been implicated in mediating
shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev.
Biochem. 57:505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev.
Immunol. 7:625-655). Furthermore, TNF.alpha. has been implicated in
the pathophysiology of a variety of other human diseases and
disorders, including sepsis, infections, autoimmune diseases,
transplant rejection and graft-versus-host disease (see e.g.,
Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A., et al. Vasilli, P. (1992) Annu. Rev.
Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev.
Med. 45:491-503).
[0002] Because of the harmful role of human TNF.alpha.
(hTNF.alpha.) in a variety of human disorders, therapeutic
strategies have been designed to inhibit or counteract hTNF.alpha.
activity. In particular, antibodies that bind to, and neutralize,
hTNF.alpha. have been sought as a means to inhibit hTNF.alpha.
activity. Some of the earliest of such antibodies were mouse
monoclonal antibodies (mAbs), secreted by hybridomas prepared from
lymphocytes of mice immunized with hTNF.alpha. (see e.g, Hahn T; et
al., (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, C-M., et
al. (1986) Biochem. Biophys. Res. Commun. 137:847-854; Hirai, M.,
et al. (1987) J. Immunol. Methods 96:57-62; Fendly, B. M., et al.
(1987) Hybridoma 6:359-370; Moeller, A., et al. (1990) Cytokine
2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European
Patent Publication No. 186 833 B1 by Wallach, D.; European Patent
Application Publication No. 218 868 A1 by Old et al.; European
Patent Publication No. 260 610 B1 by Moeller, A., et al.). While
these mouse anti-hTNF.alpha. antibodies often displayed high
affinity for hTNF.alpha. (e.g., Kd.ltoreq.10.sup.-9M) and were able
to neutralize hTNF.alpha. activity, their use in vivo may be
limited by problems associated with administration of mouse
antibodies to humans, such as short serum half life, an inability
to trigger certain human effector functions and elicitation of an
unwanted immune response against the mouse antibody in a human (the
"human anti-mouse antibody" (HAMA) reaction).
[0003] In an attempt to overcome the problems associated with use
of fully-murine antibodies in humans, murine anti-hTNF.alpha.
antibodies have been genetically engineered to be more
"human-like." For example, chimeric antibodies, in which the
variable regions of the antibody chains are murine-derived and the
constant regions of the antibody chains are human-derived, have
been prepared (Knight, D. M, et al. (1993) Mol. Immunol.
30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P. E., et
al.). Additionally, humanized antibodies, in which the
hypervariable domains of the antibody variable regions are
murine-derived but the remainder of the variable regions and the
antibody constant regions are human-derived, have also been
prepared (PCT Publication No. WO 92/11383 by Adair, J. R., et al.).
However, because these chimeric and humanized antibodies still
retain some murine sequences, they still may elicit an unwanted
immune reaction, the human anti-chimeric antibody (HACA) reaction,
especially when administered for prolonged periods, e.g., for
chronic indications, such as rheumatoid arthritis (see e.g.,
Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J.,
et al. (1994) Lancet 344:1105-1110).
[0004] A preferred hTNF.alpha. inhibitory agent to murine mAbs or
derivatives thereof (e.g., chimeric or humanized antibodies) would
be an entirely human anti-hTNF.alpha. antibody, since such an agent
should not elicit the HAMA reaction, even if used for prolonged
periods. Human monoclonal autoantibodies against hTNF.alpha. have
been prepared using human hybridoma techniques (Boyle, P., et al.
(1993) Cell. Immunol. 152:556-568; Boyle, P., et al. (1993) Cell.
Immunol. 152:569-581; European Patent Application Publication No.
614 984 A2 by Boyle, et al.). However, these hybridoma-derived
monoclonal autoantibodies were reported to have an affinity for
hTNF.alpha. that was too low to calculate by conventional methods,
were unable to bind soluble hTNF.alpha. and were unable to
neutralize hTNF.alpha.-induced cytotoxicity (see Boyle, et al.;
supra). Moreover, the success of the human hybridoma technique
depends upon the natural presence in human peripheral blood of
lymphocytes producing autoantibodies specific for hTNF.alpha..
Certain studies have detected serum autoantibodies against
hTNF.alpha. in human subjects (Fomsgaard, A., et al. (1989) Scand.
J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte
Biol. 10B:447-452), whereas others have not (Leusch, H-G., et al.
(1991) J. Immunol. Methods 139:145-147).
[0005] Alternative to naturally-occurring human anti-hTNF.alpha.
antibodies would be a recombinant hTNF.alpha. antibody. Recombinant
human antibodies that bind hTNF.alpha. with relatively low affinity
(i.e., K.sub.d.about.10.sup.-7M) and a fast off rate (i.e.,
K.sub.off.about.10.sup.-2 sec.sup.-1) have been described
(Griffiths, A. D., et al. (1993) EMBO J. 12:725-734). However,
because of their relatively fast dissociation kinetics, these
antibodies may not be suitable for therapeutic use. Additionally, a
recombinant human anti-hTNF.alpha. has been described that does not
neutralize hTNF.alpha. activity, but rather enhances binding of
hTNF.alpha. to the surface of cells and enhances internalization of
hTNF.alpha. (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45;
PCT Publication No. WO 92/03145 by Aston, R. et al.)
[0006] Recombinant human antibodies that bind soluble hTNF.alpha.
with high affinity and slow dissociation kinetics and that have the
capacity to neutralize hTNF.alpha. activity, including
hTNF.alpha.-induced cytotoxicity (in vitro and in vivo) and
hTNF.alpha.-induced cell activation, have also been described (see
U.S. Pat. No. 6,090,382).
SUMMARY OF THE INVENTION
[0007] There is a need for a stable aqueous pharmaceutical
formulation with an extended shelf life, comprising an antibody
which is suitable for therapeutic use to inhibit or counteract
detrimental hTNF.alpha. activity. There is also a need for a stable
aqueous pharmaceutical formulation with an extended shelf life,
comprising an antibody suitable for therapeutic use which is easily
administered and contains a high protein concentration.
[0008] This invention provides a liquid aqueous pharmaceutical
formulation consisting of a therapeutically effective amount of an
antibody in a buffered solution forming a formulation having a pH
between about 4 and about 8 and having a shelf life of at least 18
months. The invention also includes an aqueous pharmaceutical
formulation comprising a therapeutically effective amount of an
antibody in a buffered solution forming a formulation having a pH
between about 4 and 8 and having a shelf life of at least 18 months
in the liquid state. In one embodiment, the pharmaceutical
formulation has enhanced stability. In a further embodiment, the
formulation of the invention is stable following at least 3
freeze/thaw cycles of the formulation. In another embodiment, the
antibody is directed to TNF.alpha.. In yet another embodiment, the
antibody is directed to human TNF.alpha.. In still another
embodiment, the antibody is D2E7.
[0009] This invention also provides a liquid aqueous pharmaceutical
formulation comprising a therapeutically effective amount of an
antibody in a buffered solution forming a formulation having a pH
between 4 and 8 and having enhanced stability of at least 12 months
at a temperature of 2-8.degree. C. In one embodiment, the
formulation has enhanced stability of at least 18 months. In
another embodiment, the antibody is directed to TNF.alpha.. In yet
another embodiment, the antibody is directed to human TNF.alpha..
In a further embodiment, the antibody is D2E7.
[0010] The invention further provides a liquid aqueous
pharmaceutical formulation comprising a therapeutically effective
amount of an antibody in a buffered solution forming a formulation
having a pH between about 4 and about 8 which is easily
administratable. In one embodiment, the antibody is directed to
TNF.alpha.. In yet another embodiment, the antibody is directed to
human TNF.alpha.. In a further embodiment, the antibody is
D2E7.
[0011] In one embodiment of the invention, the liquid aqueous
pharmaceutical formulation is suitable for injection. In a further
embodiment, the formulation is suitable for single use sc
injection. In another embodiment, the concentration of the antibody
in the liquid aqueous pharmaceutical formulation is about 1-150
mg/ml. In yet another embodiment, the concentration of the antibody
in the formulation is about 50 mg/ml. In still another embodiment,
the formulation has a high protein concentration. In yet another
embodiment of the invention, the formulation is not light
sensitive.
[0012] In one embodiment of the invention, the liquid aqueous
pharmaceutical formulation contains an antibody, or an
antigen-binding portion thereof, that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less. In another embodiment,
the formulation of the invention contains an antibody, or
antigen-binding portion thereof, which dissociates from human
TNF.alpha. with a K.sub.off rate constant of 5.times.10.sup.-4
s.sup.-1 or less. In a further embodiment, the formulation contains
an antibody, or antigen-binding portion thereof, which dissociates
from human TNF.alpha. with a K.sub.off rate constant of
1.times.10.sup.-4 s.sup.-1 or less. In still a further embodiment,
the formulation of the invention contains an antibody, or
antigen-binding portion thereof, which neutralizes human TNF.alpha.
cytotoxicity in a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-8 M or less. In yet another embodiment of the
invention, the claimed formulation includes an antibody, or
antigen-binding portion thereof, which neutralizes human TNF.alpha.
cytotoxicity in a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-9 M or less. Another embodiment of the invention,
includes a formulation where the antibody, or antigen-binding
portion thereof, neutralizes human TNF.alpha. cytotoxicity in a
standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-10 M or less.
[0013] In another embodiment of the invention, the liquid aqueous
pharmaceutical formulation contains of an antibody, or
antigen-binding portion thereof, which is a recombinant antibody,
or antigen-binding portion thereof. In another embodiment, the
formulation contains an antibody, or antigen-binding portion
thereof, which inhibits human TNF.alpha.-induced expression of
ELAM-1 on human umbilical vein endothelial cells. In still another
embodiment, the claimed formulation includes the D2E7 antibody.
[0014] In another embodiment of the invention, the liquid aqueous
pharmaceutical formulation includes an antibody, or antigen-binding
portion, thereof which dissociates from human TNF.alpha. with a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as
determined by surface plasmon resonance;
[0015] b) has a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9;
[0016] c) has a heavy chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single
alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or
by one to five conservative amino acid substitutions at positions
2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12. In another embodiment, the
formulation of the invention includes an antibody, or an
antigen-binding portion thereof, which dissociates from human
TNF.alpha. with a K.sub.off rate constant of 5.times.10.sup.-4
s.sup.-1 or less. In yet another embodiment of the invention, the
formulation includes an antibody, or an antigen-binding portion
thereof, which dissociates from human TNF.alpha. with a K.sub.off
rate constant of 1.times.10.sup.-4 s.sup.-1 or less.
[0017] In yet another embodiment of the invention, the liquid
aqueous pharmaceutical formulation, contains of an antibody, or
antigen-binding portion thereof, which has a light chain variable
region (LCVR) having a CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy
chain variable region (HCVR) having a CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4
by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9,
10 or 11. In a further embodiment, the formulation of the invention
contains an antibody, wherein the LCVR of the antibody, or an
antigen-binding portion thereof, further has a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 5 and the HCVR of
the antibody, or an antigen-binding portion thereof, further has a
CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6. In
yet another embodiment, the formulation of the invention contains
an antibody, wherein the LCVR of the antibody, or an
antigen-binding portion thereof, further has CDR1 domain comprising
the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1
domain comprising the amino acid sequence of SEQ ID NO: 8.
[0018] In yet another embodiment of the invention, the antibody or
antigen-binding portion thereof, contained in the liquid aqueous
pharmaceutical formulation has a light chain variable region (LCVR)
comprising the amino acid sequence of SEQ ID NO: 1 and a heavy
chain variable region (HCVR) comprising the amino acid sequence of
SEQ ID NO: 2. In another embodiment, the antibody, or
antigen-binding portion thereof, has an IgG1 heavy chain constant
region. In still another embodiment, the antibody, or
antigen-binding portion thereof, has an IgG4 heavy chain constant
region. In another embodiment, the antibody, or antigen-binding
portion thereof, is a Fab fragment. In still a further embodiment,
the antibody, or antigen-binding portion thereof, is a single chain
Fv fragment.
[0019] In one embodiment of the invention, the liquid aqueous
pharmaceutical formulation, contains an antibody, or
antigen-binding portion thereof, which has a light chain variable
region (LCVR) having a CDR3 domain comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
SEQ ID NO: 25, SEQ ID NO: 26 or with a heavy chain variable region
(HCVR) having a CDR3 domain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. In still another
embodiment, the antibody, or antigen-binding portion thereof,
neutralizes the activity of human TNF.alpha., chimpanzee TNF.alpha.
and at least one additional primate TNF.alpha. selected from the
group consisting of baboon TNF.alpha., marmoset TNF.alpha.,
cynomolgus TNF.alpha. and rhesus TNF.alpha.. In a further
embodiment, the formulation of the invention includes an antibody,
or an antigen-binding portion thereof, which also neutralizes the
activity of mouse TNF.alpha.. The formulation of the invention also
an antibody, or an antigen-binding portion thereof, which
neutralizes the activity of pig TNF.alpha..
[0020] In a further embodiment, the invention provides a liquid
aqueous pharmaceutical formulation which contains an antibody, or
antigen-binding portion thereof, which binds to human TNF.alpha.
and comprises:
[0021] a light chain CDR3 domain comprising the amino acid sequence
of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine
substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9, and
[0022] a heavy chain CDR3 domain comprising the amino acid sequence
of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine
substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to
five conservative amino acid substitutions at positions 2, 3, 4, 5,
6, 8, 9, 10, 11 and/or 12. In one embodiment, the liquid aqueous
pharmaceutical formulation includes an antibody which bind human
TNF.alpha. and comprises a light chain variable region (LCVR)
having a CDR3 domain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26 or a heavy chain variable region (HCVR) having a CDR3
domain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID
NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33
and SEQ ID NO: 34. In a further embodiment of the invention, the
antibody, or antigen-binding portion thereof, binds human
TNF.alpha. and is the antibody D2E7 or an antigen binding portion
thereof.
[0023] The invention also provides an aqueous pharmaceutical
composition comprising a polyol, a surfactant, and a buffer system
comprising citrate and/or phosphate with a pH of about 4 to 8, in
amounts sufficient to formulate an antibody for therapeutic use at
a concentration of greater than about 45 mg/ml. In one embodiment,
the polyol is mannitol and the surfactant is polysorbate 80. In
another embodiment, the composition includes 5-20 mg/ml of mannitol
and 0.1-10 mg/ml of polysorbate 80. In a further embodiment, the
composition includes the antibody D2E7.
[0024] The invention also provides a liquid aqueous pharmaceutical
formulation consisting of 1-150 mg/ml of antibody, 5-20 mg/ml of
mannitol, 0.1-10 mg/ml of Tween-80, and a buffer system comprising
citrate and/or phosphate, with a pH of 4 to 8. In one embodiment,
the antibody is directed to hTNF.alpha.. In another embodiment, the
formulation contains about 40 mg of antibody. The invention further
provides a liquid aqueous pharmaceutical formulation comprising
about 50 mg/ml of antibody, about 12 mg/ml of mannitol, about 1
mg/ml of Tween-80, and a buffer system comprising citrate and/or
phosphate, with a pH of about 4 to about 8. In one embodiment, the
pH of the formulation is between about 4.5 to about 6.0. In another
embodiment, the pH is between about 4.8 to about 5.5. In yet
another embodiment, the pH of the invention is between about 5.0 to
about 5.2.
[0025] In one embodiment of the invention, the liquid aqueous
pharmaceutical formulation also includes about 1.305 mg/ml of
citric acid, about 0.305 mg/ml of sodium citrate, about 1.53 mg/ml
of disodium phosphate dihydrate, about 0.86 mg/ml of sodium
dihydrogen phosphate dihydrate, and about 6.165 mg/ml of sodium
chloride. In another embodiment, the formulation of the invention
includes an antibody which is directed to hTNF.alpha.. In yet
another embodiment, the formulation of the invention includes the
antibody D2E7. In yet a further embodiment, the formulation of the
invention is administered to a subject suffering from a disorder in
which TNF.alpha. activity is detrimental such that TNF.alpha.
activity in the subject is inhibited
DETAILED DESCRIPTION OF THE INVENTION
[0026] This invention pertains to a liquid aqueous pharmaceutical
formulation with a pH of about 4 to about 8 which contains a high
protein concentration, including an antibody concentration ranging
from about 1 to about 150 mg/ml, and has enhanced stability. This
invention also pertains to a liquid aqueous pharmaceutical
formulation for therapeutic use in a subject suffering from a
condition characterized by detrimental TNF.alpha. activity. The
formulation of the invention comprises the following constituents:
an antibody which binds to human TNF.alpha. with high affinity, a
low off rate and high neutralizing capacity; a buffer, which
includes citric acid, sodium citrate, disodium phosphate dihydrate,
and sodium dihydrogen phosphate dihydrate; tonicity agents, which
include mannitol and sodium chloride; a detergent, including
polysorbate 80; and sodium hydroxide, for pH adjustment.
[0027] Definitions
[0028] In order that the present invention may be more readily
understood, certain terms are first defined.
[0029] 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.
[0030] The term "pharmaceutical formulation" refers to preparations
which are in such form as to permit the biological activity of the
active ingredients to be unequivocally effective, and which contain
no additional components which are significantly toxic to the
subjects to which the formulation would be administered.
"Pharmaceutically acceptable" excipients (vehicles, additives) are
those which can reasonably be administered to a subject mammal to
provide an effective dose of the active ingredient employed.
[0031] A "stable" formulation is one in which the antibody therein
essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Various
analytical techniques for measuring protein stability are available
in the art and are reviewed in Peptide and Protein Drug Delivery,
247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,
Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 35 29-90
(1993), for example. Stability can be measured at a selected
temperature for a selected time period. Preferably, the formulation
is stable at room temperature (about 30.degree. C.) or at
40.degree. C. for at least 1 month and/or stable at about
2-8.degree. C. for at least 1 year for at least 2 years.
Furthermore, the formulation is preferably stable following
freezing (to, e.g., -70.degree. C.) and thawing of the formulation,
hereinafter referred to as a "freeze/thaw cycle."
[0032] An antibody "retains its physical stability" in a
pharmaceutical formulation if it shows substantially no signs of
aggregation, precipitation and/or denaturation upon visual
examination of color and/or clarity, or as measured by UV light
scattering or by size exclusion chromatography.
[0033] An antibody "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the antibody is considered to still retain its
biological activity as defined below. Chemical stability can be
assessed by detecting and quantifying chemically altered forms of
the antibody. Chemical alteration may involve size modification
(e.g. clipping) which can be evaluated using size exclusion
chromatography, SDS-PAGE and/or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for
example. Other types of chemical alteration include charge
alteration (e.g. occurring as a result of deamidation) which can be
evaluated by ion-exchange chromatography, for example.
[0034] An antibody "retains its biological activity" in a
pharmaceutical formulation, if the antibody in a pharmaceutical
formulation is biologically active for its intended purpose. For
example, biological activity is retained if the biological activity
of the antibody in the pharmaceutical formulation is within about
30%, about 20%, or about 10% (within the errors of the assay) of
the biological activity exhibited at the time the pharmaceutical
formulation was prepared (e.g., as determined in an antigen binding
assay).
[0035] "Isotonic" is a term recognized in the art. Isotonic can
mean, for example, that the formulation of interest has essentially
the same osmotic pressure as human blood. Isotonic formulations
will generally have an osmotic pressure from about 250 to 350 mOsm.
Isotonicity can be measured using a vapor pressure or ice-freezing
type osmometer, for example. A "tonicity agent" is a compound which
renders the formulation isotonic.
[0036] A "polyol" is a substance with multiple hydroxyl groups, and
includes sugars (reducing and nonreducing sugars), sugar alcohols
and sugar acids. Preferred polyols herein have a molecular weight
which 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. Examples of reducing sugars are fructose, mannose, maltose,
lactose, arabinose, xylose, ribose, rhamnose, galactose and
glucose. Nonreducing sugars include sucrose, trehalose, sorbose,
melezitose and raffinose. Mannitol, xylitol, erythritol, threitol,
sorbitol and glycerol are examples of sugar alcohols. As to sugar
acids, these include L-gluconate and metallic salts thereof. Where
it desired that the formulation is freeze-thaw stable, the polyol
is preferably one which does not crystallize at freezing
temperatures (e.g. -20.degree. C.) such that it destabilizes the
antibody in the formulation. The polyl may also act as a tonicity
agent. In one embodiment of the invention, one ingredient of the
formulation is mannitol in a concentration of 5 to 20 mg/ml. In a
preferred embodiment of the invention, the concentration of
mannitol is 7.5 to 15 mg/ml. In a more preferred embodiment of the
invention, the concentration of mannitol is 10-14 mg/ml.
[0037] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention has a pH in the range from
about 4 to about 8; preferably from about 4.5 to about 7; and most
preferably has a pH in the range from about 5.0 to about 6.5.
Examples of buffers that will control the pH in this range include
acetate (e.g. sodium acetate), succinate (such as sodium
succinate), gluconate, histidine, citrate and other organic acid
buffers.
[0038] In a pharmacological sense, in the context of the present
invention, a "therapeutically effective amount" or "effective
amount" of an antibody refers to an amount effective in the
prevention or treatment of a disorder for the treatment of which
the antibody is effective. A "disorder" is any condition that would
benefit from treatment with the antibody. This includes chronic and
acute disorders or diseases including those pathological conditions
which predisposes the subject to the disorder in question.
[0039] A "preservative" is a compound which can be included in the
formulation to essentially reduce bacterial action therein, thus
facilitating the production of a multi-use formulation, for
example. Examples of potential preservatives include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a mixture of alkylbenzyldimethylammonium
chlorides in which the alkyl groups are long-chain compounds), and
benzethonium chloride. Other types of preservatives include
aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl
parabens such as methyl or propyl paraben, catechol, resorcinol,
cyclohexanol, 3-pentanol, and m-cresol.
[0040] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0041] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0042] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0043] The phrase "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material,
composition or vehicle, suitable for administration to mammals. The
carriers include liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject agent from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the
patient.
[0044] The term "human TNF.alpha." (abbreviated herein as
hTNF.alpha., or simply hTNF), as used herein, is intended to refer
to a human cytokine that exists as a 17 kD secreted form and a 26
kD membrane associated form, the biologically active form of which
is composed of a trimer of noncovalently bound 17 kD molecules. The
structure of hTNF.alpha. is described further in, for example,
Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al.
(1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989)
Nature 338:225-228. The term human TNF.alpha. is intended to
include recombinant human TNF.alpha. (rhTNF.alpha.), which can be
prepared by standard recombinant expression methods or purchased
commercially (R & D Systems, Catalog No. 210-TA, Minneapolis,
Minn.).
[0045] The term "antibody", as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. The VH and VL regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the
invention, the formulation contains an antibody with CDR1, CDR2,
and CDR3 sequences like those described in U.S. Pat. Nos. 6,090,382
and 6,258,562, each incorporated by reference herein.
[0046] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., hTNF.alpha.). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CHI domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546 ), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. Other
forms of single chain antibodies, such as diabodies are also
encompassed. Diabodies are bivalent, bispecific antibodies in which
VH and VL 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. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)
Structure 2:1121-1123). In one embodiment of the invention, the
formulation contains an antigen-binding portions described in U.S.
Pat. Nos. 6,090,382 and 6,258,562, each incorporated by reference
herein.
[0047] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecules, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93- 101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein.
[0048] The term "human antibody", as used herein, 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.
[0049] The term "recombinant human antibody", as used herein, 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 (described further in Section II,
below), antibodies isolated from a recombinant, combinatorial human
antibody library (described further in Section III, below),
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.
[0050] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hTNF.alpha. is substantially free
of antibodies that specifically bind antigens other than
hTNF.alpha.. An isolated antibody that specifically binds
hTNF.alpha. may, however, have cross-reactivity to other antigens,
such as TNF.alpha. molecules from other species. Moreover, an
isolated antibody may be substantially free of other cellular
material and/or chemicals.
[0051] A "neutralizing antibody", as used herein (or an "antibody
that neutralized hTNF.alpha. activity"), is intended to refer to an
antibody whose binding to hTNF.alpha. results in inhibition of the
biological activity of hTNF.alpha.. This inhibition of the
biological activity of hTNF.alpha. can be assessed by measuring one
or more indicators of hTNF.alpha. biological activity, such as
hTNF.alpha.-induced cytotoxicity (either in vitro or in vivo),
hTNF.alpha.-induced cellular activation and hTNF.alpha. binding to
hTNF.alpha. receptors. These indicators of hTNF.alpha. biological
activity can be assessed by one or more of several standard in
vitro or in vivo assays known in the art, and described in U.S.
Pat. Nos. 6,090,382 and 6,258,562, each incorporated by reference
herein. Preferably, the ability of an antibody to neutralize
hTNF.alpha. activity is assessed by inhibition of
hTNF.alpha.-induced cytotoxicity of L929 cells. As an additional or
alternative parameter of hTNF.alpha. activity, the ability of an
antibody to inhibit hTNF.alpha.-induced expression of ELAM-1 on
HUVEC, as a measure of hTNF.alpha.-induced cellular activation, can
be assessed.
[0052] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0053] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0054] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0055] II. Antibodies of the Formulation
[0056] The invention is directed to a liquid aqueous pharmaceutical
formulation comprising a therapeutically effective amount of an
antibody in a buffered solution forming a formulation having a pH
between about 4 and about 8 and having an extended shelf life,
preferably of at least about 18 months. In another embodiment, the
liquid aqueous pharmaceutical formulation of the invention has
enhanced stability. In a further embodiment of the invention, the
formulation is not light sensitive. In yet another embodiment of
the invention, the claimed formulation remains stable following at
least 3 freeze/thaw cycles. In still another embodiment, the
pharmaceutical formulation of the invention is suitable for single
use sc injection.
[0057] Antibodies that can be used in the formulation include
polyclonal, monoclonal, recombinant antibodies, single chain
antibodies, hybrid antibodies, chimeric antibodies, humanized
antibodies, or fragments thereof. Antibody-like molecules
containing one or two binding sites for an antigen and a Fc-part of
an immunoglobulin can also be used. An example of an antibody-like
molecule is the active ingredient etanercept or infliximab.
Preferred antibodies used in the formulation are human antibodies
which are cloned from human cells or from gene-archives
representing the human antibody-reservoir. Especially preferred
among the human antibodies are antibodies directed against the
antigen TNF.alpha., including human TNF.alpha. (or
hTNF.alpha.).
[0058] In one embodiment, the formulation of the invention includes
a combination of antibodies (two or more), or a "cocktail" of
antibodies. For example, the formulation can include the antibody
D2E7 and one or more additional antibodies.
[0059] In a preferred embodiment of the invention, the formulation
contains an antibody, or antigen-binding portion thereof,
dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less and a K.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, both determined by surface
plasmon resonance, and neutralizes human TNF.alpha. cytotoxicity in
a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less. In another preferred embodiment, the
formulation of the invention contains an antibody, or
antigen-binding portion thereof, like those described in U.S. Pat.
Nos. 6,090,382 and 6,258,562, each incorporated by reference
herein.
[0060] In one aspect, the formulation of the invention contains
D2E7 antibodies and antibody portions, D2E7-related antibodies and
antibody portions, and other human antibodies and antibody portions
with equivalent properties to D2E7, such as high affinity binding
to hTNF.alpha. with low dissociation kinetics and high neutralizing
capacity. In another one embodiment, the formulation of the
invention contains an isolated human antibody, or an
antigen-binding portion thereof, that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably, the
isolated human antibody, or antigen-binding portion thereof,
dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less, or even more preferably, with a
K.sub.off of 1.times.10.sup.-4 s.sup.-1 or less. More preferably,
the isolated human antibody, or antigen-binding portion thereof,
neutralizes human TNF.alpha. cytotoxicity in a standard in vitro
L929 assay with an IC.sub.50 of 1.times.10.sup.-8 M or less, even
more preferably with an IC.sub.50 of 1.times.10.sup.-9 M or less
and still more preferably with an IC.sub.50 of 5.times.10.sup.-10 M
or less. In a preferred embodiment, the formulation contains an
antibody which is an isolated human recombinant antibody, or an
antigen-binding portion thereof. In another preferred embodiment,
the formulation contains an antibody which also neutralizes
TNF.alpha.-induced cellular activation, as assessed using a
standard in vitro assay for TNF.alpha.-induced ELAM-1 expression on
human umbilical vein endothelial cells (HUVEC).
[0061] III. Preparation of Formulation
[0062] The present invention features formulations (e.g., protein
formulations and/or antibody formulations) having improved
properties as compared to art-recognized formulations. For example,
the formulations of the invention have an improved shelf life
and/or stability as compared to art recognized formulations. In a
preferred aspect, the formulations of the invention comprise a high
protein concentration, including, for example, a protein
concentration greater than about 45 mg/ml, a protein concentration
greater than about 50 mg/ml, a protein concentration greater than
about 100 mg/ml, or a protein concentration greater than about 150
mg/ml. In a preferred embodiment of the invention, the protein is
an antibody. In another preferred embodiment, the antibody is D2E7.
The invention also provides an aqueous pharmaceutical composition
comprising a polyol, a surfactant, and a buffer system comprising
citrate and/or phosphate with a pH of about 4 to 8, in amounts
sufficient to formulate an antibody for therapeutic use at a
concentration of greater than about, for example, 45 mg/ml.
[0063] Preparation of the antibody of interest is performed
according to standard methods known in the art. In a preferred
embodiment of the invention, the antibody used in the formulation
is expressed in CHO cells and purified by a standard series of
chromatography steps. In a further preferred embodiment, the
antibody is directed to hTNF.alpha., and is prepared according to
the methods described in U.S. Pat. Nos. 6,090,382 and 6,258,562,
each incorporated by reference herein.
[0064] After preparation of the antibody of interest, the
pharmaceutical formulation comprising the antibody is prepared. The
therapeutically effective amount of antibody present in the
formulation is determined, for example, by taking into account the
desired dose volumes and mode(s) of administration. In one
embodiment of the invention, the concentration of the antibody in
the formulation is between about 1 to about 150 mg of antibody per
ml of liquid formulation. In a preferred embodiment, the
concentration of the antibody in the formulation is between about 5
to about 80 mg per ml. In another preferred embodiment, the
concentration of the antibody in the formulation is between about
25 to about 50 mg/ml. The formulation is especially suitable for
large antibody dosages of more than 15 mg/ml. In a preferred
embodiment, the concentration of the antibody is 50 mg/ml.
[0065] In another embodiment of the invention, the concentration of
the antibody in the formulation is about 1-150 mg/ml, about 5-145
mg/ml, about 10-140 mg/ml, about 15-135 mg/ml, about 20-130 mg/ml,
about 25-125 mg/ml, about 30-120 mg/ml, about 35-115 mg/ml, about
40-110 mg/ml, about 45-105 mg/ml, about 50-100 mg/ml, about 55-95
mg/ml, about 60-90 mg/ml, about 65-85 mg/ml, about 70-80 mg/ml, or
about 75 mg/ml. Ranges intermediate to the above recited
concentrations, e.g., about 6-144 mg/ml, 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.
[0066] In one embodiment, the invention provides a formulation with
an extended shelf life comprising of an active ingredient,
preferably an antibody, in combination with mannitol, citric acid
monohydrate, sodium citrate, disodium phosphate dihydrate, sodium
dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80,
water, and sodium hydroxide. In a further embodiment, the
formulation of the invention has an extended shelf life of at least
about 18 months in the liquid state. Freezing the formulation of
the invention can also be used to further extend its shelf
life.
[0067] An aqueous formulation is prepared comprising the antibody
in a pH-buffered solution. The buffer of this invention has a pH
ranging from about 4 to about 8, preferably from about 4.5 to about
6.0, more preferably from about 4.8 to about 5.5, and most
preferably has a pH of about 5.0 to about 5.2. 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. Examples of buffers that will control the
pH within this range include acetate (e.g. sodium acetate),
succinate (such as sodium succinate), gluconate, histidine, citrate
and other organic acid buffers.
[0068] In a preferred embodiment of the invention, the formulation
comprises a buffer system which contains citrate and phosphate to
maintain the pH in a range of about 4 to about 8. In a further
preferred embodiment the pH range is from about 4.5 to about 6.0,
more preferably from about pH 4.8 to about 5.5, and most preferably
in a pH range of about 5.0 to about 5.2. In another preferred
embodiment, the buffer system includes citric acid monohydrate,
sodium citrate, disodium phosphate dihydrate, and/or sodium
dihydrogen phosphate dihydrate. In a further preferred embodiment,
the buffer system includes about 1.3 mg/ml of citric acid (e.g.,
1.305 mg/ml), about 0.3 mg/ml of sodium citrate (e.g., 0.305
mg/ml), about 1.5 mg/ml of disodium phosphate dihydrate (e.g. 1.53
mg/ml), about 0.9 mg/ml of sodium dihydrogen phosphate dihydrate
(e.g., 0.86), and about 6.2 mg/ml of sodium chloride (e.g., 6.165
mg/ml). In additional preferred embodiments, the buffer system
includes 1-1.5 mg/ml of citric acid, 0.25 to 0.5 mg/ml of sodium
citrate, 1.25 to 1.75 mg/ml of of disodium phosphate dihydrate, 0.7
to 1.1 mg/ml of sodium dihydrogen phosphate dihydrate, and 6.0 to
6.4 mg/ml of sodium chloride. In a further embodiment, the pH of
the formulation is adjusted with sodium hydroxide.
[0069] A polyol, which acts as a tonicifier and may stabilize the
antibody, is also included in the formulation. The polyol is added
to the formulation in an amount which may vary with respect to the
desired isotonicity of the formulation. Preferably the aqueous
formulation is isotonic. The amount of polyol added may also alter
with respect to the molecular weight of the polyol. For example, a
lower amount of a monosaccharide (e.g. mannitol) may be added,
compared to a disaccharide (such as trehalose). In a preferred
embodiment of the invention, the polyol which is used in the
formulation as a tonicity agent is mannitol. In a preferred
embodiment of the invention, the mannitol concentration is about 5
to 20 mg/ml. In another preferred embodiment of the invention, the
concentration of mannitol is about 7.5 to 15 mg/ml. In a more
preferred embodiment of the formulation of the invention, the
concentration of mannitol is about 10-14 mg/ml. In the most
preferred embodiment, the concentration of mannitol is about 12
mg/ml. In another embodiment of the invention, the polyol sorbitol
is included in the formulation.
[0070] A detergent or surfactant is also added to the antibody
formulation. Exemplary detergents include nonionic detergents such
as polysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g.
poloxamer 188). The amount of detergent added is such that it
reduces aggregation of the formulated antibody and/or minimizes the
formation of particulates in the formulation and/or reduces
adsorption. In a preferred embodiment of the invention, the
formulation includes a surfactant which is a polysorbate. In
another preferred embodiment of the invention, the formulation
contains the detergent polysorbate 80 or Tween 80. Tween 80 is a
term used to describe polyoxyethylene (20) sorbitanmonooleate (see
Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf,
4th edi., 1996). In one preferred embodiment, the formulation
contains between about 0.1 and about 10 mg/ml of polysorbate 80,
more preferably between about 0.5 and about 5 mg/ml. In another
preferred embodiment, about 0.1% polysorbate 80 is found in the
formulation of the invention.
[0071] In a preferred embodiment of the invention, the formulation
is a 0.8 mL solution in a vial containing the ingredients shown
below in Table 1.
1TABLE 1 1 vial with 0.8 mL solution for injection.sup.1) contains:
Name of ingredient Quantity Function Active substance: Antibody
(D2E7).sup.2) 40.0 mg Active substance Excipients: Mannitol 9.6 mg
Tonicity agent Citric acid monohydrate 1.044 mg Buffer Citric acid
Sodium citrate 0.244 mg Buffer Sodium citrate Disodium phosphate
1.224 mg Buffer dihydrate Dibasic sodium phosphate dihydrate Sodium
dihydrogen 0.688 mg Buffer phosphate dihydrate Monobasic sodium
phosphate dihydrate Sodium chloride 4.932 mg Tonicity agent
Polysorbate 80 0.8 mg Detergent Water for injections
759.028-759.048 mg Solvent Water for injection Sodium
hydroxide.sup.3) 0.02-0.04 mg pH adjustment Total 817.6 mg
.sup.1)Density of the solution: 1.022 g/mL .sup.2)Is used as
concentrate .sup.3)Addition as 1 M solution
[0072] In one embodiment, the formulation contains the
above-identified agents (i.e. antibody, buffer, polyol and
detergent) and is essentially free of one or more preservatives,
such as benzyl alcohol, phenol, m-cresol, chlorobutanol and
benzethonium Cl. In another embodiment, a preservative may be
included in the formulation, particularly where the formulation is
a multidose formulation. One or more other pharmaceutically
acceptable carriers, excipients or stabilizers such as those
described in Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980) may be included in the formulation provided
that they do not significantly adversely affect the desired
characteristics of the formulation. Acceptable carriers, excipients
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed and include; additional buffering agents;
co-solvents; antioxidants including ascorbic acid and methionine;
chelating agents such as EDTA; metal complexes (e.g. Zn-protein
complexes); biodegradable polymers such as polyesters; and/or
salt-forming counterions such as sodium.
[0073] The formulation herein may also be combined with one or more
other therapeutic agents as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect the antibody of the formulation. Such
therapeutic agents are suitably present in combination in amounts
that are effective for the purpose intended. Additional therapeutic
agents which can be combined with the formulation of the invention
are further described in U.S. Pat. Nos. 6,090,382 and 6,258,562,
each of which is incorporated herein by reference.
[0074] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to, or following, preparation
of the formulation.
[0075] IV. Administration of Formulation
[0076] The formulation of the invention can be used in similar
indications as those described in U.S. Pat. Nos. 6,090,382 and
6,258,562, each incorporated by reference herein, and further
detailed below.
[0077] The language "effective amount" of the formulation is that
amount necessary or sufficient to inhibit TNF.alpha. activity,
e.g., prevent the various morphological and somatic symptoms of a
detrimental TNF.alpha. activity-associated state. In another
embodiment, the effective amount of the formulation is the amount
necessary to achieve the desired result. In one example, an
effective amount of the formulation is the amount sufficient to
inhibit detrimental TNF.alpha. activity. In another example, an
effective amount of the formulation is 0.8 mL of the formulation
containing 40 mg of antibody, as described in table 1. The
effective amount can vary depending on such factors as the size and
weight of the subject, or the type of illness. For example, the
choice of a TNF.alpha. activity-inhibiting formulation can affect
what constitutes an "effective amount". One of ordinary skill in
the art would be able to study the aforementioned factors and make
the determination regarding the effective amount of the TNF.alpha.
activity inhibiting formulation without undue experimentation.
[0078] The regimen of administration can affect what constitutes an
effective amount. The TNF.alpha. activity-inhibiting formulation
can be administered to the subject either prior to or after the
onset of detrimental TNF.alpha. activity. Further, several divided
dosages, as well as staggered dosages, can be administered daily or
sequentially, or the dose can be continuously infused, or can be a
bolus injection. Further, the dosages of the TNF.alpha.
activity-inhibiting formulation can be proportionally increased or
decreased as indicated by the exigencies of the therapeutic or
prophylactic situation.
[0079] The term "treated," "treating" or "treatment" includes the
diminishment or alleviation of at least one symptom associated or
caused by the state, disorder or disease being treated. For
example, treatment can be diminishment of one or several symptoms
of a disorder or complete eradication of a disorder.
[0080] Actual dosage levels of the active ingredients (antibody) in
the pharmaceutical formulation of this invention may be varied so
as to obtain an amount of the active ingredient that is effective
to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being
toxic to the patient.
[0081] The selected dosage level will depend upon a variety of
factors including the activity of the antibody found in the
formulation, the route of administration, the time of
administration, the rate of excretion of the particular compound
being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular
compound employed, the age, sex, weight, condition, general health
and prior medical history of the patient being treated, and like
factors well known in the medical arts.
[0082] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition of the present invention required. For
example, the physician or veterinarian could start doses of the
compounds of the invention employed in the pharmaceutical
formulation at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved.
[0083] In general, a suitable daily dose of a formulation of the
invention will be that amount of the formulation that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. An
effective amount of the formulation of the present invention is an
amount that inhibits TNF.alpha. activity in a subject suffering
from a disorder in which TNF.alpha. activity is detrimental. In a
preferred embodiment, the formulation provides an effective dose of
40 mg per injection of the active ingredient, the antibody. In
another embodiment, the formulation provides an effective dose
which ranges from about 1 to 150 mg of antibody. If desired, the
effective daily dose of the pharmaceutical formulation may be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms.
[0084] In one embodiment of the invention, the dosage of the
antibody in the formulation is between about 5 to about 80 mg. In
another embodiment, the dosage of the antibody in the formulation
is between about 25 to about 50 mg. The formulation is especially
suitable for large antibody dosages of more than 15 mg. In a
preferred embodiment of the invention, the formulation provides an
antibody at a dose of about 40 mg. In another preferred embodiment,
the antibody is directed to TNF.alpha.. In the most preferred
embodiment, the antibody is D2E7.
[0085] In one embodiment of the invention, the dosage of the
antibody in the formulation is between about 1-150 mg, about 5-145
mg, about 10-140 mg, about 15-135 mg, about 20-130 mg, about 25-125
mg, about 30-120 mg, about 35-115 mg, about 40-110 mg, about 45-105
mg, about 50-100 mg, about 55-95 mg, about 60-90 mg, about 65-85
mg, about 70-80 mg, or about 75 mg. In a preferred embodiment, the
dosage of the antibody is 40 mg. In a further preferred embodiment,
the antibody is directed to TNF.alpha.. In the most preferred
embodiment, the antibody is D2E7. Ranges intermediate to the above
recited dosages, e.g., about 2-149 mg, 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.
[0086] 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.
[0087] The invention provides a pharmaceutical formulation with an
extended shelf life, which, in one embodiment, is used to inhibit
TNF.alpha. activity in a subject suffering from a disorder in which
TNF.alpha. activity is detrimental, comprising administering to the
subject an antibody or antibody portion of the invention such that
TNF.alpha. activity in the subject is inhibited. Preferably, the
TNF.alpha. is human TNF.alpha. and the subject is a human subject.
Alternatively, the subject can be a mammal expressing a TNF.alpha.
with which an antibody of the invention cross-reacts. Still further
the subject can be a mammal into which has been introduced
hTNF.alpha. (e.g., by administration of hTNF.alpha. or by
expression of an hTNF.alpha. transgene). A formulation of the
invention can be administered to a human subject for therapeutic
purposes (discussed further below). In one embodiment of the
invention, the liquid pharmaceutical formulation is easily
administratable, which includes, for example, a formulation which
is self-administered by the patient. In a preferred embodiment, the
formulation of the invention is administered through sc injection,
preferably single use. Moreover, a formulation of the invention can
be administered to a non-human mammal expressing a TNF.alpha. with
which the antibody cross-reacts (e.g., a primate, pig or mouse) for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
[0088] As used herein, the term "a disorder in which TNF.alpha.
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF.alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF.alpha. activity is detrimental
is a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above.
[0089] There are numerous examples of disorders in which TNF.alpha.
activity is detrimental. Examples of disorders in which TNF.alpha.
activity is detrimental are described in U.S. application Ser. No.
60/397275, incorporated by reference herein. Examples in which
TNF.alpha. activity is detrimental are also described in U.S. Pat.
Nos. 6,015,557, 6,177,077, 6,379,666, 6,419,934, 6,419,944,
6,423,321, and 6,428,787; U.S. patent application Ser. Nos.
US2001/0016195, US2001/0004456, and US2001/026801; WO 00/50079 and
WO 01/49321, each incorporated by reference herein.
[0090] The use of the antibodies and antibody portions of the
invention in the treatment of specific disorders is discussed
further below:
[0091] A. Sepsis
[0092] Tumor necrosis factor has an established role in the
pathophysiology of sepsis, with biological effects that include
hypotension, myocardial suppression, vascular leakage syndrome,
organ necrosis, stimulation of the release of toxic secondary
mediators and activation of the clotting cascade (see e.g., Tracey,
K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503; Russell, D
and Thompson, R. C. (1993) Curr. Opin. Biotech. 4:714-721).
Accordingly, the formulation of the invention can be used to treat
sepsis in any of its clinical settings, including septic shock,
endotoxic shock, gram negative sepsis and toxic shock syndrome.
[0093] Furthermore, to treat sepsis, the formulation of the
invention can be coadministered with one or more additional
therapeutic agents that may further alleviate sepsis, such as an
interleukin-1 inhibitor (such as those described in PCT Publication
Nos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see
e.g., PCT Publication No. WO 93/11793) or an antagonist of platelet
activating factor (see e.g., European Patent Application
Publication No. EP 374 510).
[0094] Additionally, in a preferred embodiment, the formulation of
the invention is administered to a human subject within a subgroup
of sepsis patients having a serum or plasma concentration of IL-6
above 500 pg/ml, and more preferably 1000 pg/ml, at the time of
treatment (see PCT Publication No. WO 95/20978 by Daum, L., et
al.).
[0095] B. Autoimmune Diseases
[0096] Tumor necrosis factor has been implicated in playing a role
in the pathophysiology of a variety of autoimmune diseases. For
example, TNF.alpha. has been implicated in activating tissue
inflammation and causing joint destruction in rheumatoid arthritis
(see e.g., Tracey and Cerami, supra; Arend, W. P. and Dayer, J-M.
(1995) Arth. Rheum. 38:151-160; Fava, R. A., et al. (1993) Clin.
Exp. Immunol. 94:261-266). TNF.alpha. also has been implicated in
promoting the death of islet cells and in mediating insulin
resistance in diabetes (see e.g., Tracey and Cerami, supra; PCT
Publication No. WO 94/08609). TNF.alpha. also has been implicated
in mediating cytotoxicity to oligodendrocytes and induction of
inflammatory plaques in multiple sclerosis (see e.g., Tracey and
Cerami, supra). Chimeric and humanized murine anti-hTNF.alpha.
antibodies have undergone clinical testing for treatment of
rheumatoid arthritis (see e.g., Elliott, M. J., et al. (1994)
Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet
344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.
34:334-342).
[0097] The formulation of the invention can be used to treat
autoimmune diseases, in particular those associated with
inflammation, including rheumatoid arthritis, rheumatoid
spondylitis, osteoarthritis and gouty arthritis, allergy, multiple
sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic
syndrome. Typically, the formulation is administered systemically,
although for certain disorders, local administration of the
antibody or antibody portion at a site of inflammation may be
beneficial (e.g., local administration in the joints in rheumatoid
arthritis or topical application to diabetic ulcers, alone or in
combination with a cyclohexane-ylidene derivative as described in
PCT Publication No. WO 93/19751).
[0098] C. Infectious Diseases
[0099] Tumor necrosis factor has been implicated in mediating
biological effects observed in a variety of infectious diseases.
For example, TNF.alpha. has been implicated in mediating brain
inflammation and capillary thrombosis and infarction in malaria
(see e.g., Tracey and Cerami, supra). TNF.alpha. also has been
implicated in mediating brain inflammation, inducing breakdown of
the blood-brain barrier, triggering septic shock syndrome and
activating venous infarction in meningitis (see e.g., Tracey and
Cerami, supra). TNF.alpha. also has been implicated in inducing
cachexia, stimulating viral proliferation and mediating central
nervous system injury in acquired immune deficiency syndrome (AIDS)
(see e.g., Tracey and Cerami, supra). Accordingly, the antibodies,
and antibody portions, of the invention, can be used in the
treatment of infectious diseases, including bacterial meningitis
(see e.g., European Patent Application Publication No. EP 585 705),
cerebral malaria, AIDS and AIDS-related complex (ARC) (see e.g.,
European Patent Application Publication No. EP 230 574), as well as
cytomegalovirus infection secondary to transplantation (see e.g.,
Fietze, E., et al. (1994) Transplantation 58:675-680). The
formulation of the invention, also can be used to alleviate
symptoms associated with infectious diseases, including fever and
myalgias due to infection (such as influenza) and cachexia
secondary to infection (e.g., secondary to AIDS or ARC).
[0100] D. Transplantation
[0101] Tumor necrosis factor has been implicated as a key mediator
of allograft rejection and graft versus host disease (GVHD) and in
mediating an adverse reaction that has been observed when the rat
antibody OKT3, directed against the T cell receptor CD3 complex, is
used to inhibit rejection of renal transplants (see e.g., Tracey
and Cerami, supra; Eason, J. D., et al. (1995) Transplantation
59:300-305; Suthanthiran, M. and Strom, T. B. (1994) New Engl. J.
Med. 331:365-375). Accordingly, the formulation of the invention,
can be used to inhibit transplant rejection, including rejections
of allografts and xenografts and to inhibit GVHD. Although the
antibody or antibody portion may be used alone, more preferably it
is used in combination with one or more other agents that inhibit
the immune response against the allograft or inhibit GVHD. For
example, in one embodiment, the formulation of the invention is
used in combination with OKT3 to inhibit OKT3-induced reactions. In
another embodiment, the formulation of the invention is used in
combination with one or more antibodies directed at other targets
involved in regulating immune responses, such as the cell surface
molecules CD25 (interleukin-2 receptor-.alpha.), CD11a (LFA-1),
CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86
(B7-2). In yet another embodiment, the formulation of the invention
is used in combination with one or more general immunosuppressive
agents, such as cyclosporin A or FK506.
[0102] E. Malignancy
[0103] Tumor necrosis factor has been implicated in inducing
cachexia, stimulating tumor growth, enhancing metastatic potential
and mediating cytotoxicity in malignancies (see e.g., Tracey and
Cerami, supra). Accordingly, the formulation of the invention, can
be used in the treatment of malignancies, to inhibit tumor growth
or metastasis and/or to alleviate cachexia secondary to malignancy.
The formulation may be administered systemically or locally to the
tumor site.
[0104] F. Pulmonary Disorders
[0105] Tumor necrosis factor has been implicated in the
pathophysiology of adult respiratory distress syndrome, including
stimulating leukocyte-endothelial activation, directing
cytotoxicity to pneumocytes and inducing vascular leakage syndrome
(see e.g., Tracey and Cerami, supra). Accordingly, the formulation
of the invention, can be used to treat various pulmonary disorders,
including adult respiratory distress syndrome (see e.g., PCT
Publication No. WO 91/04054), shock lung, chronic pulmonary
inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and
silicosis. The formulation may be administered systemically or
locally to the lung surface, for example as an aerosol.
[0106] G. Intestinal Disorders
[0107] Tumor necrosis factor has been implicated in the
pathophysiology of inflammatory bowel disorders (see e.g., Tracy,
K. J., et al. (1986) Science 234:470-474; Sun, X-M., et al. (1988)
J. Clin. Invest. 81:1328-1331; MacDonald, T. T., et al. (1990)
Clin. Exp. Immunol. 81:301-305). Chimeric murine anti-hTNF.alpha.
antibodies have undergone clinical testing for treatment of Crohn's
disease (van Dullemen, H. M., et al. (1995) Gastroenterology
109:129-135). The formulation of the invention, also can be used to
treat intestinal disorders, such as idiopathic inflammatory bowel
disease, which includes two syndromes, Crohn's disease and
ulcerative colitis.
[0108] H. Cardiac Disorders
[0109] The formulation of the invention, also can be used to treat
various cardiac disorders, including ischemia of the heart (see
e.g., European Patent Application Publication No. EP 453 898) and
heart insufficiency (weakness of the heart muscle) (see e.g., PCT
Publication No. WO 94/20139).
[0110] I. Others
[0111] The pharmaceutical formulation of the invention, also can be
used to treat various other disorders in which TNF.alpha. activity
is detrimental. Examples of other diseases and disorders in which
TNF.alpha. activity has been implicated in the pathophysiology, and
thus which can be treated using the formulation of the invention,
include inflammatory bone disorders and bone resorption disease
(see e.g., Bertolini, D. R., et al. (1986) Nature 319:516-518;
Konig, A., et al. (1988) J. Bone Miner. Res. 3:621-627; Lerner, U.
H. and Ohlin, A. (1993) J. Bone Miner. Res. 8:147-155; and Shankar,
G. and Stern, P. H. (1993) Bone 14:871-876), hepatitis, including
alcoholic hepatitis (see e.g., McClain, C. J. and Cohen, D. A.
(1989) Hepatology 9:349-35 1; Felver, M. E., et al. (1990) Alcohol.
Clin. Exp. Res. 14:255-259; and Hansen, J., et al. (1994)
Hepatology 20:461-474) and viral hepatitis (Sheron, N., et al.
(1991) J. Hepatol. 12:241-245; and Hussain, M. J., et al. (1994) J.
Clin. Pathol. 47:1112-1115), coagulation disturbances (see e.g.,
van der Poll, T., et al. (1990) N. Engl. J. Med. 322:1622-1627; and
van der Poll, T., et al. (1991) Prog. Clin. Biol. Res. 367:55-60),
burns (see e.g., Giroir, B. P., et al. (1994) Am. J. Physiol.
267:H1 18-124; and Liu, X. S., et al. (1994) Burns 20:40-44),
reperfusion injury (see e.g., Scales, W. E., et al. (1994) Am. J.
Physiol. 267:G1122-1127; Serrick, C., et al. (1994) Transplantation
58:1158-1162; and Yao, Y. M., et al. (1995) Resuscitation
29:157-168), keloid formation (see e.g., McCauley, R. L., et al.
(1992) J. Clin. Immunol. 12:300-308), scar tissue formation;
pyrexia; periodontal disease; obesity and radiation toxicity.
[0112] Other disorders in which TNF.alpha. activity is detrimental
include, but are not limited to, adult Still's disease, Alzheimer's
disease, ankylosing spondylitis, asthma, cancer and cachexia,
atherosclerosis, chronic atherosclerosis, chronic fatigue syndrome,
liver failure, chronic liver failure, obstructive pulmonary
disease, chronic obstructive pulmonary disease, congestive heart
failure, dermatopolymyositis, diabetic macrovasculopathy,
endometriosis, familial periodic fevers, fibrosis, hemodialysis,
Jarisch-Herxheimer reaction, juvenile RA, Kawasaki syndrome, myelo
dysplastic syndrome, myocardial infarction, panciaticular vulgaris,
periodontal disease, peripheral neuropathy, polyarticular,
polymyositis, progressive renal failure, psoriasis, psoriatic
arthritis, Reiter's syndrome, sarcoidosis, scleroderma,
spondyloarthropathies, Still's disease, stroke, therapy associated
syndrome, therapy induced inflammatory syndrome, inflammatory
syndrome following IL-2 administration, thoracoabdominal aortic
aneurysm repair (TAAA), Vasulo-Behcet's disease, Yellow Fever
vaccination, type 1 diabetes mellitus, type 2 diabetes mellitus,
neuropathic pain, sciatica, cerebral edema, edema in and/or around
the spinal cord, vasculitide, Wegener's granulomatosis, temporal
arteritis, polymyalgia rheumatica, Takayasu's arteritis,
polyarteritis nodosa, microscopic polyangiitis, Churg-Strauss
syndrome, Felty's syndrome, Sjogren's syndrome, mixed connective
tissue disorder, relapsing polychondritis, pseudogout, loosening of
prostheses, autoimmune hepatitis, sclerosing cholangitis, acute
pancreatitis, chronic pancreatitis, glomerulonephritides,
post-streptococcal glomerulonephritis or IgA nephropathy, rheumatic
heart disease, cardiomyopathy, orchitis, pyoderma gangerenosum,
multiple myeloma, TNF receptor associated periodic syndrome
[TRAPS], atherosclerosis, steroid dependent giant cell
arteritismyostitis, uveitis, and drug reactions.
[0113] The invention is further illustrated in the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
Example 1
Preparation of the Formulation
[0114] The pharmaceutical formulation of the invention was made
according to the following protocol.
[0115] Materials which were used in the formulation include:
mannitol, citric acid monohydrate (citric acid), sodium citrate,
disodium phosphate dihydrate (dibasic sodium phosphate dihydrate),
sodium dihydrogen phosphate dihydrate (monobasic sodium phosphate
dihydrate), sodium chloride, polysorbate 80, water for the
injections, sodium hydroxide, which was used as a 1M solution to
adjust the pH, and protein concentrate (e.g., antibody
concentrate).
[0116] Preparation of 20L of Buffer (Equivalent to 20.180
kg--Density of the Solution: 1.009 g/ml
[0117] Ingredients were weighed out as follows: 240.0 g mannitol,
26.1 g citric acid monohydrate, 6.1 g sodium citrate, 30.6 g
disodium phosphate dihydrate, 17.2 g sodium dihydrogen phosphate
dihydrate, 123.3 g sodium chloride, 20.0 g polysorbate 80, and
19,715.7 to 19,716.1 g of water.
[0118] A sodium hydroxide solution was prepared by combining 40.0 g
of sodium hydroxide with 1000.8 g of water for injections.
[0119] Next, a buffer was prepared by dissolving the following
pre-weighed ingredients (described above) in about 90% of the water
for injections: mannitol, citric acid monohydrate, sodium citrate,
disodium phosphate dihydrate, sodium dihydrogen phosphate, sodium
chloride, and polysorbate 80. It was determined that the sequence
of the addition of the buffer constituents was not important and
can, therefore, be chosen at will.
[0120] Following addition of all of the buffer constituents, the pH
of the solution was adjusted with 1M sodium hydroxide which was
prepared as described above. After the addition of the sodium
hydroxide, the final weight of the water was added. The buffer
solution was then filtered through a sterilized filter (hydrophilic
polyvinylidene difluoride, 0.22 .mu.m pore size) into a sterilized
receptacle. The filtration medium used was filtration sterilized
nitrogen.
[0121] Preparation of 40L of Formulation (Equivalent to 40.88
kg)
[0122] The filtered buffer solution was then added to the thawed
and pooled antibody concentrate (the active ingredient of the
pharmaceutical formulation), prepared as follows. The antibody
(concentrate) was thawed in a water bath prior to the preparation
of the pharmaceutical formulation. 34.207 g of antibody concentrate
was used, which is equivalent to 2.0 kg of protein with 60 mg
protein/mL protein concentrate. The density of the concentrate was
1.0262 g/mL. Any protein concentrate ranging from 25.655 to 37.316,
which is equivalent to a protein concentration in the protein
concentrate of 55 to 80 mg/mL, can be used. The buffer was added
while stirring, until the final weight of the bulk solution was
reached.
[0123] The formulation, with all of its ingredients included, was
then sterilized by filtration as described above, except the
formulation was filtered through two sterile 0.22 .mu.m membrane
filters. Following sterilization, the formulation was packaged for
use in either a vial or a pre-filled syringe.
[0124] The skilled artisan will also appreciate that the weight
quantities and/or weight-to-volume ratios recited herein, can be
converted to moles and/or molarities using the art-recognized
molecular weights of the recited ingredients. Weight quantities
exemplified herein (e.g., g or kg) are for the volumes (e.g., of
buffer or pharmaceutical formulation) recited. The skilled artisan
will appreciate that the weight quantities can be proportionally
adjusted when different formulation volumes are desired. For
example, 32L, 20L, 10L, 5L, or 1L formulations would include 80%,
50%, 25%, 12.5%, or 2.5%, respectively, of the exemplified weight
quantities.
Example 2
Freeze/Thaw Studies
[0125] After the formulation buffer for the D2E7 antibody was
selected the drug substance was formulated in the same matrix as
the finished product.
[0126] Freeze thaw behavior of the D2E7 antibody drug substance at
a protein concentration of 63 mg/mL was evaluated by cycling drug
substance 3 times from the frozen state to the liquid state. Table
N shows the results of an experiment evaluating the effect of three
fast and slow freeze-thaw cycles in the presence and absence of
0.1% polysorbate 80 starting from -80.degree. C. or -30.degree. C.,
respectively.
[0127] Table 2 shows that the D2E7 antibody drug substance can be
thawed/frozen at least 3 times without any detrimental effect on
either chemical (cation exchange HPLC, size exclusion HPLC, colour,
pH), physicochemical properties (subvisible particles, clarity) or
biological activity (in vitro TNF neutralization assay). Also table
2 shows that the inclusion of polysorbate 80 improved the
physicochemical properties of the D2E7 antibody drug substance as
evidenced by the lower number of subvisible particles regardless
whether a slow or fast freeze/thaw cycle was being used (see shaded
areas in table 2).
2TABLE 2 Effect of freeze thaw on the D2E7 antibody drug substance
with/without polysorbate 80 Fast Fast thaw thaw Poly- No Slow thaw
-30.degree. C. in Slow thaw -80.degree. C. in sorbate freeze/
-30.degree. C. in water -80.degree. in water Test criteria
(0.1%).sup.1) thaw refrigerator bath refrigerator bath Clarity -
25.0 22.5 25.3 25.8 25.6 + 27.8 28.1 28.2 28.0 28.1 Colour -
.ltoreq.B9 .ltoreq.B9 .ltoreq.B9 .ltoreq.B9 .ltoreq.B9 + .ltoreq.B9
.ltoreq.B9 .ltoreq.B9 .ltoreq.B9 .ltoreq.B9 pH - 5.01 5.02 5.02
5.02 5.02 + 5.02 5.02 5.02 5.02 5.02 Subvisible - 42 600 303 1891
303 particles 2 4 5 8 0 + 0 5 1 0 8 0 0 0 0 1 Size - 99.8 99.8 99.8
99.8 99.8 exclusion HPLC + 99.8 99.8 99.8 99.8 99.8 Cation - 87.1
87.0 87.2 86.9 86.9 exchange + 86.8 87.0 87.1 87.3 86.8 HPLC In
vitro TNF - 118.0 123.8 118.0 103.3 120.5 neutrali- + 111.8 96.2
100.9 96.7 95.8 zation test .sup.1)+ = formulation with 0.1%
polysorbate 80; - = formulation without 0.1% polysorbate 80
Example 3
Microbial Studies
[0128] Tests were performed to determine if the formulation can
support microbial growth. The results from these experiments showed
that the formulation does not support microbial growth if stored at
20 to 25.degree. C. for 14 days. This result was determined by
directly inoculating the sterile formulation with microorganisms
(e.g., Staphylococous aureus, ATCC-No.: 6538P, Candida albicans,
ATCC-No.: 10231, Aspergillus niger, ATCCC-No.: 16404, Pseudomonas
aeruginosa, ATCC-No.: 9027, an environmental isolate) at low level
(NMT 100 cfu/mL). Inoculated formulations were then examined for
overall microbial growth, e.g., for changes in turbidity. A lack of
turbidity was an indication of no overall growth, and was detected
in the inoculated containers after 14 days. Further, no organisms
could be reisolated from these containers. Thus it was concluded
that the formulation does not support microbial growth under these
conditions.
[0129] Incorporation by Reference
[0130] The contents of all references and patents cited herein are
hereby incorporated by reference in their entirety.
[0131] Equivalents
[0132] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 0
0
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