U.S. patent application number 13/288299 was filed with the patent office on 2012-07-12 for methods of administering anti-tnfalpha antibodies.
This patent application is currently assigned to ABBOTT BIOTECHNOLOGY LTD.. Invention is credited to Steven A. Fischkoff, Joachim Kempeni, Roberta Weiss.
Application Number | 20120177596 13/288299 |
Document ID | / |
Family ID | 23144283 |
Filed Date | 2012-07-12 |
United States Patent
Application |
20120177596 |
Kind Code |
A1 |
Fischkoff; Steven A. ; et
al. |
July 12, 2012 |
METHODS OF ADMINISTERING ANTI-TNFalpha ANTIBODIES
Abstract
Methods of treating disorders in which TFN.alpha. activity is
detrimental via biweekly, subcutaneous administration of human
antibodies, preferably recombinant human antibodies, that
specifically bind to human tumor necrosis factor .alpha.
(hTNF.alpha.) are disclosed. The antibody may be administered with
or without methotrexate. These antibodies have high affinity for
hTNF.alpha. (e.g., K.sub.d=10.sup.-8 M or less), a slow off rate
for hTNF.alpha. dissociation (e.g., K.sub.off=10.sup.-3 sec.sup.-1
or less) and neutralize hTNF.alpha. activity in vitro and in vivo.
An antibody of the invention can be a full-length antibody or an
antigen-binding portion thereof. Kits containing a pharmaceutical
composition and instructions for dosing, and preloaded syringes
containing pharmaceutical compositions are also encompassed by the
invention.
Inventors: |
Fischkoff; Steven A.; (Short
Hills, NJ) ; Kempeni; Joachim; (Neustadt, DE)
; Weiss; Roberta; (Wynnewood, PA) |
Assignee: |
ABBOTT BIOTECHNOLOGY LTD.
HAMILTON
BM
|
Family ID: |
23144283 |
Appl. No.: |
13/288299 |
Filed: |
November 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10163657 |
Jun 5, 2002 |
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13288299 |
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60296961 |
Jun 8, 2001 |
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Current U.S.
Class: |
424/85.2 ;
424/135.1; 424/158.1 |
Current CPC
Class: |
A61P 37/06 20180101;
C07K 2317/21 20130101; A61P 31/04 20180101; A61P 31/14 20180101;
A61K 2039/545 20130101; A61P 27/02 20180101; A61P 3/10 20180101;
A61P 19/02 20180101; A61P 29/02 20180101; A61K 2300/00 20130101;
A61P 1/04 20180101; A61P 43/00 20180101; A61P 31/20 20180101; A61P
19/08 20180101; C07K 2317/52 20130101; A61P 3/00 20180101; A61P
17/02 20180101; A61P 37/02 20180101; A61K 39/3955 20130101; A61P
7/04 20180101; A61K 2039/54 20130101; A61P 25/00 20180101; C07K
2317/565 20130101; A61P 11/00 20180101; A61P 27/00 20180101; A61P
1/00 20180101; A61P 9/10 20180101; A61P 13/12 20180101; A61P 19/10
20180101; A61K 31/519 20130101; A61P 7/00 20180101; A61K 9/0019
20130101; A61P 37/00 20180101; Y02A 50/30 20180101; A61P 13/00
20180101; Y02A 50/412 20180101; C07K 2317/76 20130101; A61P 19/00
20180101; A61P 31/00 20180101; A61P 37/08 20180101; A61P 17/00
20180101; A61P 41/00 20180101; A61P 9/00 20180101; C07K 2317/56
20130101; A61P 1/16 20180101; A61P 29/00 20180101; A61P 35/00
20180101; C07K 16/241 20130101; A61K 2039/505 20130101; A61P 31/12
20180101; A61P 19/06 20180101; A61K 39/3955 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/85.2 ;
424/158.1; 424/135.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 1/00 20060101 A61P001/00; A61P 1/04 20060101
A61P001/04; A61K 38/20 20060101 A61K038/20 |
Claims
1. A method for treating an intestinal disorder in a human subject,
comprising subcutaneously administering a total body dose of 20-80
mg of an isolated human anti-TNF.alpha. antibody, or an
antigen-binding fragment thereof, to the human subject, on a
biweekly dosing regimen such that the intestinal disorder is
treated.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein said human antibody, or an
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.
5. The method of claim 4, wherein said human antibody, or
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.off rate constant of 5.times.10.sup.-4 s.sup.-1 or
less.
6. The method of claim 4, wherein said human antibody, or
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or
less.
7. The method of claim 4, wherein said 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.
8. The method of claim 4, wherein said 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.-9 M or less.
9. The method of claim 4, wherein said 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.-10 M or less.
10. (canceled)
11. A method for inhibiting human TNF.alpha. activity in a human
subject suffering from an intestinal disorder, comprising
subcutaneously administering a total body dose of 20-80 mg of an
isolated human anti-TNF.alpha. antibody, or an antigen-binding
fragment thereof, to the human subject on a biweekly dosing
regimen, wherein said human antibody, or antigen-binding portion
thereof, has the following characteristics: 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,
dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less as 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.
12. (canceled)
13. The method of claim 11, wherein said human antibody, or an
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.off rate constant of 5.times.10.sup.-4 s.sup.-1 or
less.
14. A method for inhibiting human TNF.alpha. activity in a human
subject suffering from an intestinal disorder, comprising
subcutaneously administering a total body dose of 20-80 mg of a
human anti-TNF.alpha. antibody, or an antigen-binding fragment
thereof, to the human subject on a biweekly dosing regimen, wherein
said human antibody, or an antigen-binding portion thereof, 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,
a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5,
and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:
7, and has 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, a CDR2 domain comprising
the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain
comprising the amino acid sequence of SEQ ID NO: 8.
15. (canceled)
16. (canceled)
17. A method for inhibiting human TNF.alpha. activity in a human
subject suffering from an intestinal disorder, comprising
subcutaneously administering a total body dose of 20-80 mg of a
human anti-TNF.alpha. antibody, or an antigen-binding fragment
thereof, to the human subject on a biweekly dosing regimen, wherein
said human antibody, or an 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.
18. The method of claim 17, wherein said human antibody has an IgG1
heavy chain constant region.
19. The method of claim 17, wherein said human antibody has an IgG4
heavy chain constant region.
20. The method of claim 17, wherein said human antibody is a Fab
fragment.
21. The method of claim 17, wherein said human antibody is a single
chain Fv fragment.
22. (canceled)
23. A method for inhibiting human TNF.alpha. activity in a human
subject suffering from an intestinal disorder, comprising
subcutaneously administering a total body dose of 20-80 mg of a
human anti-TNF.alpha. antibody, or an antigen-binding fragment
thereof, to the human subject on a biweekly dosing regimen, said
composition containing a human anti-TNF.alpha. antibody wherein
said human antibody is the antibody D2E7 or an antigen-binding
portion thereof.
24.-58. (canceled)
59. The method of claim 1, wherein the antibody is administered to
the human subject together with the cytokine interleukin-6 (IL-6)
or is administered to a human subject with a serum or plasma
concentration of IL-6 above 500 pg/ml.
60.-72. (canceled)
73. The method of claim 1, wherein the intestinal disorder is an
idiopathic inflammatory bowel disease.
74. The method of claim 11, wherein the intestinal disorder is an
idiopathic inflammatory bowel disease.
75. The method of claim 14, wherein the intestinal disorder is an
idiopathic inflammatory bowel disease.
76. The method of claim 17, wherein the intestinal disorder is an
idiopathic inflammatory bowel disease.
77. The method of claim 23, wherein the intestinal disorder is an
idiopathic inflammatory bowel disease.
78. The method of claim 73, wherein the idiopathic inflammatory
bowel disease is either Crohn's disease or ulcerative colitis.
79. The method of claim 74, wherein the idiopathic inflammatory
bowel disease is either Crohn's disease or ulcerative colitis.
80. The method of claim 75, wherein the idiopathic inflammatory
bowel disease is either Crohn's disease or ulcerative colitis.
81. The method of claim 76, wherein the idiopathic inflammatory
bowel disease is either Crohn's disease or ulcerative colitis.
82. The method of claim 77, wherein the idiopathic inflammatory
bowel disease is either Crohn's disease or ulcerative colitis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/163,657, filed on Jun. 5, 2002, which claims the benefit of
the filing date under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application No. 60/296,961, filed on Jun. 8, 2001. The entire
contents of each of the foregoing applications, including
specification, any drawings, and sequence listing, are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.,
Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J.
and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).
[0003] 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; Moller, 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).
[0004] 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).
[0005] 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).
[0006] 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.)
[0007] 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). Typical protocols for administering
antibodies are performed intravenously on a weekly basis. Weekly
dosing with antibodies and/or any drug can be costly, cumbersome,
and result in an increase in the number of side effects due to the
frequency of administration. Intravenous administration also has
limitations in that the administration is usually provided by
someone with medical training.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods for biweekly dosing
regimens for the treatment of TNF.alpha. associated disorders,
preferably via a subcutaneous route. Biweekly dosing has many
advantages over weekly dosing including, but not limited to, a
lower number of total injections, decreased number of injection
site reactions (e.g., local pain and swelling), increased patient
compliance (i.e., due to less frequent injections), and less cost
to the patient as well as the health care provider. Subcutaneous
dosing is advantageous because the patient may self-administer a
therapeutic substance, e.g., a human TNF.alpha. antibody, which is
convenient for both the patient and the health care provider.
[0009] This invention provides methods for treating disorders in
which TNF.alpha. activity is detrimental. The methods include
administering biweekly, subcutaneous injections of antibodies to a
subject. The antibodies preferably are recombinant human antibodies
that specifically bind to human TNF.alpha.. This invention further
provides methods for treating disorders in which TNF.alpha.
activity is detrimental. These methods include utilizing a
combination therapy wherein human antibodies are administered to a
subject with another therapeutic agent, such as one or more
additional antibodies that bind other targets (e.g., antibodies
that bind other cytokines or that bind cell surface molecules), one
or more cytokines, soluble TNF.alpha. receptor (see e.g., PCT
Publication No. WO 94/06476) and/or one or more chemical agents
that inhibit hTNF.alpha. production or activity (such as
cyclohexane-ylidene derivatives as described in PCT Publication No.
WO 93/19751), preferably methotrexate. The antibodies are
preferably recombinant human antibodies that specifically bind to
human TNF.alpha.. The antibodies of the invention are characterized
by binding to hTNF.alpha. with high affinity and slow dissociation
kinetics and by neutralizing hTNF.alpha. activity, including
hTNF.alpha.-induced cytotoxicity (in vitro and in vivo) and
hTNF.alpha.-induced cellular activation. The antibodies can be
full-length (e.g., an IgG1 or IgG4 antibody) or can comprise only
an antigen-binding portion (e.g., a Fab, F(ab').sub.2, scFv
fragment or single domain). The most preferred recombinant antibody
of the invention, termed D2E7, has a light chain CDR3 domain
comprising the amino acid sequence of SEQ ID NO: 3 and a heavy
chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:
4 (set forth in Appendix B). Preferably, the D2E7 antibody 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. These
antibodies are described in U.S. Pat. No. 6,090,382, incorporated
in its entirety herein by reference.
[0010] In one embodiment, the invention provides methods of
treating disorders in which TNF.alpha. activity is detrimental.
These methods include inhibiting human TNF.alpha. activity by
subcutaneous, biweekly administration of an anti-TNF.alpha.
antibody such that the disorder is treated. The disorder can be,
for example, sepsis, an autoimmune disease (e.g., rheumatoid
arthritis, allergy, multiple sclerosis, autoimmune diabetes,
autoimmune uveitis and nephrotic syndrome), an infectious disease,
a malignancy, transplant rejection or graft-versus-host disease, a
pulmonary disorder, a bone disorder, an intestinal disorder or a
cardiac disorder.
[0011] In another embodiment, the invention provides methods of
treating disorders in which TNF.alpha. activity is detrimental.
These methods include inhibiting human TNF.alpha. activity by
subcutaneous administration of an anti-TNF.alpha. antibody and
methotrexate such that the disorder is treated. In one aspect,
methotrexate is administered together with an anti-TNF.alpha.
antibody. In another aspect, methotrexate is administered prior to
the administration of an anti-TNF.alpha. antibody. In still another
aspect, methotrexate is administered subsequent to the
administration of an anti-TNF.alpha. antibody.
[0012] In a preferred embodiment, the anti-TNF.alpha. antibody used
to treat disorders in which TNF.alpha. activity is detrimental is a
human anti-TNF.alpha. antibody. Even more preferably, treatment
occurs by the biweekly, subcutaneous administration of an isolated
human antibody, or an antigen-binding portion thereof. The antibody
or antigen-binding portion thereof preferably 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.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 1.times.10.sup.-10 M
or less.
[0013] In another embodiment, the invention provides methods of
treating disorders in which TNF.alpha. activity is detrimental by
the biweekly, subcutaneous administration to the subject a human
antibody, or antigen-binding portion thereof. The antibody or
antigen-binding portion thereof preferably has the following
characteristics:
[0014] a) dissociates from human TNF.alpha. with a K.sub.off 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
[0016] 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;
[0017] 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.
[0018] More preferably, the antibody, or antigen-binding portion
thereof, dissociates from human TNF.alpha. with a K.sub.off of
5.times.s.sup.-1 or less. Still more preferably, the antibody, or
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.off of 1.times.10.sup.-4 s.sup.-1 or less.
[0019] In yet another embodiment, the invention provides methods of
treating disorders in which TNF.alpha. activity is detrimental.
These methods include a biweekly, subcutaneous administration to
the subject a human antibody, or an antigen-binding portion
thereof. The antibody or antigen-binding portion thereof preferably
contains an LCVR having 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 an 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. More
preferably, the LCVR further has a CDR2 domain comprising the amino
acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2
domain comprising the amino acid sequence of SEQ ID NO: 6. Still
more preferably, the LCVR 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.
[0020] In still another embodiment, the invention provides methods
of treating disorders in which TNF.alpha. activity is detrimental
by subcutaneously administering to the subject, biweekly, an
isolated human antibody, or an antigen binding portion thereof. The
antibody or antigen-binding portion thereof preferably contains an
LCVR comprising the amino acid sequence of SEQ ID NO: 1 and an HCVR
comprising the amino acid sequence of SEQ ID NO: 2. In certain
embodiments, the antibody has an IgG1 heavy chain constant region
or an IgG4 heavy chain constant region. In yet other embodiments,
the antibody is a Fab fragment, an F(ab').sub.2 fragment or a
single chain Fv fragment.
[0021] In still other embodiments, the invention provides methods
of treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial by subcutaneously
administering to the subject, biweekly, one or more anti-TNF.alpha.
antibodies, or antigen-binding portions thereof. The antibody or
antigen-binding portion thereof preferably contains an LCVR having
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 an 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, SEQ ID NO: 34 and SEQ ID NO: 35.
[0022] Still another aspect of the invention pertains to kits
containing a formulation comprising a pharmaceutical composition.
The kits comprise an anti-TNF.alpha. antibody and a
pharmaceutically acceptable carrier. The kits contain instructions
for biweekly subcutaneous dosing of the pharmaceutical composition
for the treatment of a disorder in which the administration of an
anti-TNF.alpha. antibody is beneficial. In another aspect, the
invention pertains to kits containing a formulation comprising a
pharmaceutical composition, further comprising an anti-TNF.alpha.
antibody, methotrexate, and a pharmaceutically acceptable carrier.
The kits contain instructions for subcutaneous dosing of the
pharmaceutical composition for the treatment of a disorder in which
the administration of an anti-TNF.alpha. antibody is
beneficial.
[0023] Still another aspect of the invention provides a preloaded
syringe containing a pharmaceutical composition comprising an
anti-TNF.alpha. antibody and a pharmaceutically acceptable carrier.
In still another aspect, the invention provides a preloaded syringe
containing a pharmaceutical composition comprising an
anti-TNF.alpha. antibody, methotrexate, and a pharmaceutically
acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A and 1B depict the American College of Rheumatology
20 (ACR20) and ACR50 responses for patients suffering from
rheumatoid arthritis (RA) after subcutaneous dosing with the
antibody D2E7 every week for a total of twelve weeks (1A), or
subcutaneous dosing with the antibody D2E7 and methotrexate every
other week (1B) for a total of twenty-four weeks. These data
indicate that every other week dosing is as effective as every week
dosing.
[0025] FIG. 2 depicts ACR20, ACR50, and ACR70 responses for
patients suffering from RA after subcutaneous dosing with the
antibody D2E7 and methotrexate every other week at twenty-four
weeks.
[0026] FIGS. 3A and 3B depict time courses of tender joint count
(3A) and swollen joint count (3B) over twenty-four weeks for
patients suffering from RA after subcutaneous dosing with D2E7 and
methotrexate every other week at twenty-four weeks.
[0027] FIG. 4 depicts results from a short form health survey
(SF-36) from patients suffering from RA after subcutaneous dosing
with the antibody D2E7 and methotrexate every other week at
twenty-four weeks. RP, role physical; PF, physical function; BP,
bodily pain; GH, general health; V, vitality; SF, social
functioning; RE, role emotional; and ME, mental health.
[0028] FIG. 5 depicts the percentage of ACR responders following a
single intravenous injection of the antibody D2E7 and methotrexate
in patients suffering from RA.
DETAILED DESCRIPTION OF THE INVENTION
[0029] This invention pertains to methods of treating disorders in
which the administration of an anti-TNF.alpha. antibody is
beneficial comprising the administration of isolated human
antibodies, or antigen-binding portions thereof, that bind to human
TNF.alpha. with high affinity, a low off rate and high neutralizing
capacity such that the disorder is treated. Various aspects of the
invention relate to treatment with antibodies and antibody
fragments, and pharmaceutical compositions thereof.
[0030] In order that the present invention may be more readily
understood, certain terms are first defined.
[0031] The term "dosing", as used herein, refers to the
administration of a substance (e.g., an anti-TNF.alpha. antibody)
to achieve a therapeutic objective (e.g., the treatment of a
TNF.alpha.-associated disorder).
[0032] The terms "biweekly dosing regimen", "biweekly dosing", and
"biweekly administration", as used herein, refer to the time course
of administering a substance (e.g., an anti-TNF.alpha. antibody) to
a subject to achieve a therapeutic objective (e.g., the treatment
of a TNF.alpha.-associated disorder). The biweekly dosing regimen
is not intended to include a weekly dosing regimen. Preferably, the
substance is administered every 9-19 days, more preferably, every
11-17 days, even more preferably, every 13-15 days, and most
preferably, every 14 days.
[0033] The term "combination therapy", as used herein, refers to
the administration of two or more therapeutic substances, e.g., an
anti-TNF.alpha. antibody and the drug methotrexate. The
methotrexate may be administered concomitant with, prior to, or
following the administration of an anti-TNF.alpha. antibody.
[0034] 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 TNF.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.).
[0035] 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.
[0036] 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 CH1 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 VII 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).
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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 hTNF.alpha. molecules from other species (discussed in
further detail below). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0041] 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 (see Example 4).
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.
[0042] 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 Example 1 and
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.
[0043] 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.
[0044] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0045] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0046] The term "isolated nucleic acid molecule", as used herein in
reference to nucleic acids encoding antibodies or antibody portions
(e.g., VH, VL, CDR3) that bind hTNF.alpha., is intended to refer to
a nucleic acid molecule in which the nucleotide sequences encoding
the antibody or antibody portion are free of other nucleotide
sequences encoding antibodies or antibody portions that bind
antigens other than hTNF.alpha., which other sequences may
naturally flank the nucleic acid in human genomic DNA. Thus, for
example, an isolated nucleic acid of the invention encoding a VH
region of an anti-hTNF.alpha. antibody contains no other sequences
encoding other VH regions that bind antigens other than
hTNF.alpha..
[0047] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0048] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0049] Various aspects of the invention are described in further
detail in the following subsections.
I. Human Antibodies that Bind Human TNF.alpha.
[0050] This invention provides methods of treating disorders in
which the administration of an anti-TNF.alpha. antibody is
beneficial. These methods include the biweekly, subcutaneous
administration of isolated human antibodies, or antigen-binding
portions thereof, that bind to human TNF.alpha. with high affinity,
a low off rate and high neutralizing capacity. Preferably, the
human antibodies of the invention are recombinant, neutralizing
human anti-hTNF.alpha. antibodies. The most preferred recombinant,
neutralizing antibody of the invention is referred to herein as
D2E7 (the amino acid sequence of the D2E7 VL region is shown in SEQ
ID NO: 1; the amino acid sequence of the D2E7 VH region is shown in
SEQ ID NO: 2). The properties of D2E7 have been described in
Salfeld et al., U.S. Pat. No. 6,090,382, which is incorporated by
reference herein.
[0051] In one aspect, the invention pertains to treating disorders
in which the administration of an anti-TNF.alpha. antibody is
beneficial. These treatments include the biweekly, subcutaneous
administration of 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 one
embodiment, the invention provides treatment with 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 1.times.10.sup.-10 M
or less. In a preferred embodiment, the antibody is an isolated
human recombinant antibody, or an antigen-binding portion
thereof.
[0052] It is well known in the art that antibody heavy and light
chain CDR3 domains play an important role in the binding
specificity/affinity of an antibody for an antigen. Accordingly, in
another aspect, the invention pertains to methods of treating
disorders in which the administration of an anti-TNF.alpha.
antibody is beneficial by subcutaneous administration of human
antibodies that have slow dissociation kinetics for association
with hTNF.alpha. and that have light and heavy chain CDR3 domains
that structurally are identical to or related to those of D2E7.
Position 9 of the D2E7 VL CDR3 can be occupied by Ala or Thr
without substantially affecting the K.sub.off. Accordingly, a
consensus motif for the D2E7 VL CDR3 comprises the amino acid
sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Additionally,
position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn,
without substantially affecting the K.sub.off. Accordingly, a
consensus motif for the D2E7 VL CDR3 comprises the amino acid
sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as
demonstrated in Example 2, the CDR3 domain of the D2E7 heavy and
light chains is amenable to substitution with a single alanine
residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at
position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without
substantially affecting the K.sub.off. Still further, the skilled
artisan will appreciate that, given the amenability of the D2E7 VL
and VH CDR3 domains to substitutions by alanine, substitution of
other amino acids within the CDR3 domains may be possible while
still retaining the low off rate constant of the antibody, in
particular substitutions with conservative amino acids. A
"conservative amino acid substitution", as used herein, is one in
which one amino acid residue is replaced with another amino acid
residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art,
including basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Preferably, no
more than one to five conservative amino acid substitutions are
made within the D2E7 VL and/or VH CDR3 domains. More preferably, no
more than one to three conservative amino acid substitutions are
made within the D2E7 VL and/or VH CDR3 domains. Additionally,
conservative amino acid substitutions should not be made at amino
acid positions critical for binding to hTNF.alpha.. Positions 2 and
5 of the D2E7 VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3
appear to be critical for interaction with hTNF.alpha. and thus,
conservative amino acid substitutions preferably are not made at
these positions (although an alanine substitution at position 5 of
the D2E7 VL CDR3 is acceptable, as described above) (see U.S. Pat.
No. 6,090,382).
[0053] Accordingly, in another embodiment, the invention provides
methods of treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial by the biweekly,
subcutaneous administration of an isolated human antibody, or
antigen-binding portion thereof. The antibody or antigen-binding
portion thereof preferably contains the following
characteristics:
[0054] 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;
[0055] 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;
[0056] 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.
[0057] More preferably, the 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. Even more preferably, the
antibody, or antigen-binding portion thereof, dissociates from
human TNF.alpha. with a K.sub.off of 1.times.10.sup.-4 s.sup.-1 or
less.
[0058] In yet another embodiment, the invention provides methods of
treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial by the biweekly,
subcutaneous administration of an isolated human antibody, or an
antigen-binding portion thereof. The antibody or antigen-binding
portion thereof preferably contains 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. Preferably, the LCVR further has a CDR2 domain comprising the
amino acid sequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and
the HCVR further has a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). Even more
preferably, the LCVR further has CDR1 domain comprising the amino
acid sequence of SEQ ID NO: 7 (i.e., the D2E7 VL CDR1) and the HCVR
has a CDR1 domain comprising the amino acid sequence of SEQ ID NO:
8 (i.e., the D2E7 VH CDR1). The framework regions for VL preferably
are from the V.sub..kappa.I human germline family, more preferably
from the A20 human germline Vk gene and most preferably from the
D2E7 VL framework sequences shown in FIGS. 1A and 1B of U.S. Pat.
No. 6,090,382. The framework regions for VH preferably are from the
V.sub.H3 human germline family, more preferably from the DP-31
human germline VH gene and most preferably from the D2E7 VH
framework sequences shown in FIGS. 2A and 2B U.S. Pat. No.
6,090,382.
[0059] In still another embodiment, the invention provides methods
of treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial by the biweekly,
subcutaneous administration of an isolated human antibody, or an
antigen binding portion thereof. The antibody or antigen-binding
portion thereof preferably contains a light chain variable region
(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (i.e.,
the D2E7 VL) and a heavy chain variable region (HCVR) comprising
the amino acid sequence of SEQ ID NO: 2 (i.e., the D2E7 VH). In
certain embodiments, the antibody comprises a heavy chain constant
region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD
constant region. Preferably, the heavy chain constant region is an
IgG1 heavy chain constant region or an IgG4 heavy chain constant
region. Furthermore, the antibody can comprise a light chain
constant region, either a kappa light chain constant region or a
lambda light chain constant region. Preferably, the antibody
comprises a kappa light chain constant region. Alternatively, the
antibody portion can be, for example, a Fab fragment or a single
chain Fv fragment.
[0060] In still other embodiments, the invention provides methods
of treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial by the biweekly,
subcutaneous administration of an isolated human antibody, or an
antigen-binding portions thereof. The antibody or antigen-binding
portion thereof preferably contains D2E7-related VL and VH CDR3
domains, for example, antibodies, or antigen-binding portions
thereof, with 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 and 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,
SEQ ID NO: 34 and SEQ ID NO: 35.
[0061] An antibody or antibody portion of the invention can be
derivatized or linked to another functional molecule (e.g., another
peptide or protein). Accordingly, the antibodies and antibody
portions of the invention are intended to include derivatized and
otherwise modified forms of the human anti-hTNF.alpha. antibodies
described herein, including immunoadhesion molecules. For example,
an antibody or antibody portion of the invention can be
functionally linked (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
associate of the antibody or antibody portion with another molecule
(such as a streptavidin core region or a polyhistidine tag).
[0062] One type of derivatized antibody is produced by crosslinking
two or more antibodies (of the same type or of different types,
e.g., to create bispecific antibodies). Suitable crosslinkers
include those that are heterobifunctional, having two distinctly
reactive groups separated by an appropriate spacer (e.g.,
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional
(e.g., disuccinimidyl suberate). Such linkers are available from
Pierce Chemical Company, Rockford, Ill.
[0063] Useful detectable agents with which an antibody or antibody
portion of the invention may be derivatized include fluorescent
compounds. Exemplary fluorescent detectable agents include
fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
II. Expression of Antibodies
[0064] An antibody, or antibody portion, of the invention can be
prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. To express an antibody
recombinantly, a host cell is transfected with one or more
recombinant expression vectors carrying DNA fragments encoding the
immunoglobulin light and heavy chains of the antibody such that the
light and heavy chains are expressed in the host cell and,
preferably, secreted into the medium in which the host cells are
cultured, from which medium the antibodies can be recovered.
Standard recombinant DNA methodologies are used to obtain antibody
heavy and light chain genes, incorporate these genes into
recombinant expression vectors and introduce the vectors into host
cells, such as those described in Sambrook, Fritsch and Maniatis
(eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current
Protocols in Molecular Biology, Greene Publishing Associates,
(1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
[0065] To express D2E7 or a D2E7-related antibody, DNA fragments
encoding the light and heavy chain variable regions are first
obtained. These DNAs can be obtained by amplification and
modification of germline light and heavy chain variable sequences
using the polymerase chain reaction (PCR). Germline DNA sequences
for human heavy and light chain variable region genes are known in
the art (see e.g., the "Vbase" human germline sequence database;
see also Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M.,
et al. (1992) "The Repertoire of Human Germline V.sub.H Sequences
Reveals about Fifty Groups of V.sub.H Segments with Different
Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, J. P. L.
et al. (1994) "A Directory of Human Germ-line V.sub.78 Segments
Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836;
the contents of each of which are expressly incorporated herein by
reference). To obtain a DNA fragment encoding the heavy chain
variable region of D2E7, or a D2E7-related antibody, a member of
the V.sub.H3 family of human germline VH genes is amplified by
standard PCR. Most preferably, the DP-31 VH germline sequence is
amplified. To obtain a DNA fragment encoding the light chain
variable region of D2E7, or a D2E7-related antibody, a member of
the V.sub..kappa.I family of human germline VL genes is amplified
by standard PCR. Most preferably, the A20 VL germline sequence is
amplified. PCR primers suitable for use in amplifying the DP-31
germline VH and A20 germline VL sequences can be designed based on
the nucleotide sequences disclosed in the references cited supra,
using standard methods.
[0066] Once the germline VH and VL fragments are obtained, these
sequences can be mutated to encode the D2E7 or D2E7-related amino
acid sequences disclosed herein. The amino acid sequences encoded
by the germline VH and VL DNA sequences are first compared to the
D2E7 or D2E7-related VH and VL amino acid sequences to identify
amino acid residues in the D2E7 or D2E7-related sequence that
differ from germline. Then, the appropriate nucleotides of the
germline DNA sequences are mutated such that the mutated germline
sequence encodes the D2E7 or D2E7-related amino acid sequence,
using the genetic code to determine which nucleotide changes should
be made. Mutagenesis of the germline sequences is carried out by
standard methods, such as PCR-mediated mutagenesis (in which the
mutated nucleotides are incorporated into the PCR primers such that
the PCR product contains the mutations) or site-directed
mutagenesis.
[0067] Once DNA fragments encoding D2E7 or D2E7-related VH and VL
segments are obtained (by amplification and mutagenesis of germline
VH and VL genes, as described above), these DNA fragments can be
further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes or to a scFv gene. In
these manipulations, a VL- or VH-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked", as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0068] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH.sub.1, CH.sub.2 and CH.sub.3). The sequences
of human heavy chain constant region genes are known in the art
(see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the VH-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH1 constant
region.
[0069] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a kappa constant
region.
[0070] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., Nature (1990)
348:552-554).
[0071] To express the antibodies, or antibody portions of the
invention, DNAs encoding partial or full-length light and heavy
chains, obtained as described above, are inserted into expression
vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). Prior to insertion of the D2E7 or D2E7-related
light or heavy chain sequences, the expression vector may already
carry antibody constant region sequences. For example, one approach
to converting the D2E7 or D2E7-related VH and VL sequences to
full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is
operatively linked to the CH segment(s) within the vector and the
VL segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0072] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al.
[0073] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0074] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0075] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0076] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hTNF.alpha.. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the invention. In addition, bifunctional
antibodies may be produced in which one heavy and one light chain
are an antibody of the invention and the other heavy and light
chain are specific for an antigen other than hTNF.alpha. by
crosslinking an antibody of the invention to a second antibody by
standard chemical crosslinking methods.
[0077] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are culture to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture
medium.
III Selection of Recombinant Human Antibodies
[0078] Recombinant human antibodies of the invention in addition to
D2E7 or an antigen binding portion thereof, or D2E7-related
antibodies disclosed herein can be isolated by screening of a
recombinant combinatorial antibody library, preferably a scFv phage
display library, prepared using human VL and VH cDNAs prepared from
mRNA derived from human lymphocytes. Methodologies for preparing
and screening such libraries are known in the art. In addition to
commercially available kits for generating phage display libraries
(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SwiZAP.TM. phage display kit,
catalog no. 240612), examples of methods and reagents particularly
amenable for use in generating and screening antibody display
libraries can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et
al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication
No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;
Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al.
PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No.
WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al., Nature (1990)
348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et
al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS
88:7978-7982.
[0079] In a preferred embodiment, to isolate human antibodies with
high affinity and a low off rate constant for hTNF.alpha., a murine
anti-hTNF.alpha. antibody having high affinity and a low off rate
constant for hTNF.alpha. (e.g., MAK 195, the hybridoma for which
has deposit number ECACC 87 050801) is first used to select human
heavy and light chain sequences having similar binding activity
toward hTNF.alpha., using the epitope imprinting methods described
in Hoogenboom et al., PCT Publication No. WO 93/06213. The antibody
libraries used in this method are preferably scFv libraries
prepared and screened as described in McCafferty et al., PCT
Publication No. WO 92/01047, McCafferty et al., Nature (1990)
348:552-554; and Griffiths et al., (1993) EMBO J. 12:725-734. The
scFv antibody libraries preferably are screened using recombinant
human TNF.alpha. as the antigen.
[0080] Once initial human VL and VH segments are selected, "mix and
match" experiments, in which different pairs of the initially
selected VL and VH segments are screened for hTNF.alpha. binding,
are performed to select preferred VL/VH pair combinations.
Additionally, to further improve the affinity and/or lower the off
rate constant for hTNF.alpha. binding, the VL and VH segments of
the preferred VL/VH pair(s) can be randomly mutated, preferably
within the CDR3 region of VH and/or VL, in a process analogous to
the in vivo somatic mutation process responsible for affinity
maturation of antibodies during a natural immune response. This in
vitro affinity maturation can be accomplished by amplifying VH and
VL regions using PCR primers complimentary to the VH CDR3 or VL
CDR3, respectively, which primers have been "spiked" with a random
mixture of the four nucleotide bases at certain positions such that
the resultant PCR products encode VH and VL segments into which
random mutations have been introduced into the VH and/or VL CDR3
regions. These randomly mutated VH and VL segments can be
rescreened for binding to hTNF.alpha. and sequences that exhibit
high affinity and a low off rate for hTNF.alpha. binding can be
selected.
[0081] Following screening and isolation of an anti-hTNF.alpha.
antibody of the invention from a recombinant immunoglobulin display
library, nucleic acid encoding the selected antibody can be
recovered from the display package (e.g., from the phage genome)
and subcloned into other expression vectors by standard recombinant
DNA techniques. If desired, the nucleic acid can be further
manipulated to create other antibody forms of the invention (e.g.,
linked to nucleic acid encoding additional immunoglobulin domains,
such as additional constant regions). To express a recombinant
human antibody isolated by screening of a combinatorial library,
the DNA encoding the antibody is cloned into a recombinant
expression vector and introduced into a mammalian host cells, as
described in further detail in Section II above.
IV. Pharmaceutical Compositions and Pharmaceutical
Administration
[0082] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject for the methods described herein, e.g.,
biweekly, subcutaneous dosing. Typically, the pharmaceutical
composition comprises an antibody (or antibody portion) of the
invention and/or methotrexate and a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible
and are suitable for administration to a subject for the methods
described herein. Examples of pharmaceutically acceptable carriers
include one or more of water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol and the like, as well as combinations
thereof. In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody or antibody portion.
[0083] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular injection. In a particularly
preferred embodiment, the antibody is administered by subcutaneous
injection (e.g., a biweekly, subcutaneous injection).
[0084] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying that yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0085] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
[0086] In certain embodiments, the active compound may be prepared
with a carrier that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyethylene glycol (PEG), polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Many methods for the preparation of such formulations are patented
or generally known to those skilled in the art. See, e.g.,
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0087] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0088] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents. For example, an
anti-hTNF.alpha. antibody or antibody portion of the invention may
be coformulated and/or coadministered with methotrexate, one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules), one or more cytokines, soluble TNF.alpha. receptor (see
e.g., PCT Publication No. WO 94/06476) and/or one or more chemical
agents that inhibit hTNF.alpha. production or activity (such as
cyclohexane-ylidene derivatives as described in PCT Publication No.
WO 93/19751). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies. The use of the antibodies, or antibody
portions, of the invention in combination with other therapeutic
agents is discussed further in subsection IV.
[0089] Non-limiting examples of therapeutic agents for rheumatoid
arthritis with which an antibody, or antibody portion, of the
invention can be combined include the following: non-steroidal
anti-inflammatory drug(s) (NSAIDs); cytokine suppressive
anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized
anti-TNF.alpha. antibody; Celltech/Bayer); cA2 (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF
receptor-IgG fusion protein; Immunex; see e.g., Arthritis &
Rheumatism (1994) Vol. 37, 5295; J. Invest. Med. (1996) Vol. 44,
235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized
anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis &
Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2
(IL-2 fusion proteins; Seragen see e.g., Arthritis & Rheumatism
(1993) Vol. 36, 1223); Anti-Tac (humanized anti-IL-2R.alpha.;
Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine;
DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10,
anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4
agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor
antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding
protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), 5284; Amer. J. Physiol.--Heart and Circulatory
Physiology (1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase
Type IV inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S81); Iloprost (see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S282) and thalidomide-related drugs (e.g., Celgen); leflunomide
(anti-inflammatory and cytokine inhibitor; see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;
Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid
(inhibitor of plasminogen activation; see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S284); T-614
(cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996)
Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g.,
Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam
(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal
anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory
drug); Diclofenac (non-steroidal anti-inflammatory drug);
Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine
(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S281); Azathioprine (see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor
(inhibitor of the enzyme interleukin-1.beta. converting enzyme);
zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase
zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos
of vascular endothelial cell growth factor or vascular endothelial
cell growth factor receptor; inhibitors of angiogenesis);
corticosteroid anti-inflammatory drugs (e.g., SB203580);
TNF-convertase inhibitors; anti-IL-12 antibodies; interleukin-11
(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S296); interleukin-13 (see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S308);
interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism
(1996) Vol. 39, No. 9 (supplement), S120); gold; penicillamine;
chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide;
cyclosporine; total lymphoid irradiation; anti-thymocyte globulin;
anti-CD4 antibodies; CD5-toxins; orally-administered peptides and
collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs)
HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense
phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis
Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell
Sciences, Inc.); prednisone; orgotein; glycosaminoglycan
polysulphate; minocycline; anti-IL2R antibodies; marine and
botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca
et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin;
phenylbutazone; meclofenamic acid; flufenamic acid; intravenous
immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus
(FK-506); sirolimus (rapamycin); amiprilose (therafectin);
cladribine (2-chlorodeoxyadenosine); and azaribine.
[0090] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which an antibody, or antibody portion, of the
invention can be combined include the following: budenoside;
epidermal growth factor; corticosteroids; cyclosporin,
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor
antagonists; anti-IL-1.beta. monoclonal antibodies; anti-IL-6
monoclonal antibodies; growth factors; elastase inhibitors;
pyridinyl-imidazole compounds; CDP-571/BAY-10-3356 (humanized
anti-TNF.alpha. antibody; Celltech/Bayer); cA2 (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF
receptor-IgG fusion protein; Immunex; see e.g., Arthritis &
Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44,
235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough);
IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies);
interleukin-11; glucuronide- or dextran-conjugated prodrugs of
prednisolone, dexamethasone or budesonide; ICAM-1 antisense
phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis
Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell
Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists
of Platelet Activating Factor (PAF); ciprofloxacin; and
lignocaine.
[0091] Nonlimiting examples of therapeutic agents for multiple
sclerosis with which an antibody, or antibody portion, of the
invention can be combined include the following: corticosteroids;
prednisolone; methylprednisolone; azathioprine; cyclophosphamide;
cyclosporine; methotrexate; 4-aminopyridine; tizanidine;
interferon-.beta.1a (Avonex.TM.; Biogen); interferon-.beta.1b
(Betaseron.TM.; Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone.TM.;
Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;
intravenous immunoglobulin; clabribine; CDP-571/BAY-10-3356
(humanized anti-TNF.alpha. antibody; Celltech/Bayer); cA2 (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF
receptor-IgG fusion protein; Immunex; see e.g., Arthritis &
Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44,
235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); IL-10; IL-4; and IL-10 and/or IL-4 agonists
(e.g., agonist antibodies).
[0092] Nonlimiting examples of therapeutic agents for sepsis with
which an antibody, or antibody portion, of the invention can be
combined include the following: hypertonic saline solutions;
antibiotics; intravenous gamma globulin; continuous hemofiltration;
carbapenems (e.g., meropenem); antagonists of cytokines such as
TNF.alpha.c, IL-1.beta., IL-6 and/or IL-8; CDP-571/BAY-10-3356
(humanized anti-TNF.alpha. antibody; Celltech/Bayer); cA2 (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF
receptor-IgG fusion protein; Immunex; see e.g., Arthritis &
Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44,
235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and
HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low
molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone
CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor (TPA;
Chiron); PHP (chemically modified hemoglobin; APEX Bioscience);
iron chelators and chelates, including diethylenetriamine
pentaacetic acid-iron (III) complex (DTPA iron (III); Molichem
Medicines); lisofylline (synthetic small molecule methylxanthine;
Cell Therapeutics, Inc.); PGG-Glucan (aqeuous soluble
.beta.1,3glucan; Alpha-Beta Technology); apolipoprotein A-1
reconstituted with lipids; chiral hydroxamic acids (synthetic
antibacterials that inhibit lipid A biosynthesis); anti-endotoxin
antibodies; E5531 (synthetic lipid A antagonist; Eisai America,
Inc.); rBPI.sub.21 (recombinant N-terminal fragment of human
Bactericidal/Permeability-Increasing Protein); and Synthetic
Anti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);
[0093] Nonlimiting examples of therapeutic agents for adult
respiratory distress syndrome (ARDS) with which an antibody, or
antibody portion, of the invention can be combined include the
following: anti-IL-8 antibodies; surfactant replacement therapy;
CDP-571/BAY-10-3356 (humanized anti-TNF.alpha. antibody;
Celltech/Bayer); cA2 (chimeric anti-TNF.alpha. antibody; Centocor);
75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see
e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest.
Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNF
receptor-IgG fusion protein; Hoffmann-LaRoche).
[0094] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody or antibody portion are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0095] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0096] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 10-100 mg, more preferably 20-80 mg and
most preferably about 40 mg. It is to be noted that dosage values
may vary with the type and 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.
V. Uses of the Antibodies of the Invention
[0097] Given their ability to bind to hTNF.alpha., the
anti-hTNF.alpha. antibodies, or portions thereof, of the invention
can be used to detect hTNF.alpha. (e.g., in a biological sample,
such as serum or plasma), using a conventional immunoassay, such as
an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay
(RIA) or tissue immunohistochemistry. The invention provides a
method for detecting hTNF.alpha. in a biological sample comprising
contacting a biological sample with an antibody, or antibody
portion, of the invention and detecting either the antibody (or
antibody portion) bound to hTNF.alpha. or unbound antibody (or
antibody portion), to thereby detect hTNF.alpha. in the biological
sample. The antibody is directly or indirectly labeled with a
detectable substance to facilitate detection of the bound or
unbound antibody. Suitable detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0098] Alternative to labeling the antibody, hTNF.alpha. can be
assayed in biological fluids by a competition immunoassay utilizing
rhTNF.alpha. standards labeled with a detectable substance and an
unlabeled anti-hTNF.alpha. antibody. In this assay, the biological
sample, the labeled rhTNF.alpha. standards and the anti-hTNF.alpha.
antibody are combined and the amount of labeled rhTNF.alpha.
standard bound to the unlabeled antibody is determined. The amount
of hTNF.alpha. in the biological sample is inversely proportional
to the amount of labeled hTNF.alpha. standard bound to the
anti-hTNF.alpha. antibody.
[0099] A D2E7 antibody of the invention can also be used to detect
TNF.alpha.s from species other than humans, in particular
TNF.alpha.s from primates (e.g., chimpanzee, baboon, marmoset,
cynomolgus and rhesus), pig and mouse, since D2E7 can bind to each
of these TNF.alpha.s.
[0100] The antibodies and antibody portions of the invention are
capable of neutralizing hTNF.alpha. activity both in vitro and in
vivo (see U.S. Pat. No. 6,090,382). Moreover, at least some of the
antibodies of the invention, such as D2E7, can neutralize
hTNF.alpha. activity from other species. Accordingly, the
antibodies and antibody portions of the invention can be used to
inhibit hTNF.alpha. activity, e.g., in a cell culture containing
hTNF.alpha., in human subjects or in other mammalian subjects
having TNF.alpha.s with which an antibody of the invention
cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and
rhesus, pig or mouse). In one embodiment, the invention provides a
method for inhibiting TNF.alpha. activity comprising contacting
TNF.alpha. with an antibody or antibody portion of the invention
such that TNF.alpha. activity is inhibited. Preferably, the
TNF.alpha. is human TNF.alpha.. For example, in a cell culture
containing, or suspected of containing TNF.alpha., an antibody or
antibody portion of the invention can be added to the culture
medium to inhibit hTNF.alpha. activity in the culture.
[0101] In a preferred embodiment, the invention provides methods of
treating disorders in which the administration of an
anti-TNF.alpha. antibody is beneficial, comprising subcutaneously
administering to the subject biweekly an antibody or antibody
portion of the invention such that the disorder is treated. In a
particularly preferred embodiment, the antibody is administered
subcutaneously on a biweekly schedule. In another particularly
preferred embodiment, the antibody is administered subcutaneously
before, during or after administration of methotrexate. Preferably,
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). An
antibody of the invention can be administered to a human subject
for therapeutic purposes (discussed further below). Moreover, an
antibody 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).
[0102] As used herein, the term "a disorder in which the
administration of an anti-TNF.alpha. antibody is beneficial" 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, or where it has been shown that
another anti-TNF.alpha. antibody or a biologically active portion
thereof has been successfully used to treat the disease.
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. There are numerous
examples of disorders in which TNF.alpha. activity is detrimental.
The use of the antibodies and antibody portions of the invention in
the treatment of specific disorders is discussed further below:
[0103] A. Sepsis
[0104] 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 human antibodies, and antibody portions, 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.
[0105] Furthermore, to treat sepsis, an anti-hTNF.alpha. antibody,
or antibody portion, 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).
[0106] Additionally, in a preferred embodiment, an anti-TNF.alpha.
antibody or antibody portion 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.).
[0107] B. Autoimmune Diseases
[0108] 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, (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).
[0109] The human antibodies, and antibody portions 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 antibody, or
antibody portion, 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).
[0110] C. Infectious Diseases
[0111] 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
antibodies, and antibody portions, 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).
[0112] D. Transplantation
[0113] 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 antibodies, and antibody
portions, 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, an antibody or
antibody portion of the invention is used in combination with OKT3
to inhibit OKT3-induced reactions. In another embodiment, an
antibody or antibody portion 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, an antibody or antibody portion
of the invention is used in combination with one or more general
immunosuppressive agents, such as cyclosporin A or FK506.
[0114] E. Malignancy
[0115] 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 antibodies, and antibody portions,
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 antibody, or antibody portion, may be
administered systemically or locally to the tumor site.
[0116] F. Pulmonary Disorders
[0117] 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 antibodies,
and antibody portions, 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 antibody, or antibody
portion, may be administered systemically or locally to the lung
surface, for example as an aerosol.
[0118] G. Intestinal Disorders
[0119] 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 human antibodies, and antibody portions, 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.
[0120] H. Cardiac Disorders
[0121] The antibodies, and antibody portions, 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).
[0122] I. Others
[0123] The antibodies, and antibody portions, 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 an antibody,
or antibody portion, 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 Stem, P. H. (1993) Bone
14:871-876), hepatitis, including alcoholic hepatitis (see e.g.,
McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351;
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)
Frog. Clin. Biol. Res. 367:55-60), burns (see e.g., Giroir, B. P.,
et al. (1994) Am. J. Physiol. 267:H118-124; and Liu, X. S., et al.
(1994) Burns 20:40-44), reperfusion injury (see e.g., Scales, W.
E., et al. (1994) Am. I 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 and pyrexia.
[0124] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
Example 1
Treatment with an Anti-TNF.alpha. Antibody
D2E7 Efficacy Following Subcutaneous Administration
[0125] In this study, twenty-four patients with active RA were
treated with weekly doses of 0.5 mg/kg D2E7 (n=18) or placebo (n=6)
by s.c. injection for three months. Patients participating in this
study had a mean duration of disease of 10.1 years with a disease
activity score (DAS) score of 4.87 and a mean of 3.4 DMARDs
(disease modifying anti-rheumatic drugs) prior to study entry;
again reflecting considerable disease activity. Responders
continued open-label treatment with D2E7, while patients who failed
to respond to the 0.5 mg/kg dose or who lost a DAS response on the
0.5 mg/kg dose were escalated to receive 1 mg/kg by s.c. injection
after week twelve of the study.
[0126] The first patients enrolled received up to sixty injections
and were, therefore, sixty weeks on the study drug. The efficacy
with s.c. dosing was similar to i.v. injections. Up to 78% of
patients reached a DAS and ACR20 response during the first weeks of
treatment.
[0127] Subcutaneous D2E7 at a dose of 0.5 mg/kg/week reduced the
swollen joint (SWJ) count by 54%, tender joint count (TJC) by 61%
and CRP by 39% over twelve weeks compared to baseline, whereas all
parameters increased in the placebo group. After completion of the
placebo-controlled period of this study, the patients continued
treatment for up to fourteen months with sustained efficacy. These
results indicate that subcutaneous D2E7 at a dose of 0.5 mg/kg/week
can, therefore, be safely self-administered with good local
tolerability.
Administration of D2E7 and Methotrexate
[0128] In this study, patients received s.c. or i.v. placebo or
D2E7 at a dose of 1 mg/kg in addition to their ongoing treatment
with (methotrexate) MTX. Fifty-four patients were enrolled in the
study and eighteen patients received i.v. D2E7 and s.c. placebo,
eighteen patients received i.v. placebo and s.c. D2E7, and eighteen
patients received placebo i.v. and s.c. The patients received their
second dose only after they lost their blinded response status, not
earlier than four weeks after the first dose. Thereafter, all
patients received open-label biweekly s.c. injections of D2E7.
[0129] Demographic characteristics of the study population of this
study included a mean duration of RA of 11.1 years, prior exposure
to a mean of 3.6 DMARDs (other than MTX), and a mean DAS at study
entry of 4.81. By Day twenty-nine, 72% of the i.v. D2E7 treated
patients and 44% of the s.c. D2E7 treated patients had achieved a
response by DAS criteria, compared to only 28% of placebo-treated
patients (set forth in FIG. 5). Of the responders in this study,
28% of placebo treated patients maintained an ACR20 response up to
day 29, compared to 72% of i.v.-treated D2E7 patients and 67% of
s.c.-treated D2E7 patients, who maintained their responses for
between one and three months.
Example 2
Total Body Dose of a Subcutaneously Administered Anti-TNF.alpha.
Antibody
Weekly, Subcutaneous Administration of D2E7
[0130] This study enrolled two hundred eighty-four patients with RA
and was designed to determine the optimal total body dose of
subcutaneously administered D2E7. Patients were randomized to
receive either 20, 40, or 80 mg D2E7 or placebo weekly for twelve
weeks, after which time placebo-treated patients were switched
blindly to 40 mg D2E7/week.
[0131] Approximately 49% of patients reached ACR20 at 20 mg, 55% of
patients reached ACR20 at 40 mg, and 54% of patients reached ACR20
at 80 mg, while only 10% of patients receiving placebo reached
ACR20 (set forth in FIG. 1A). Approximately 23% of patients reached
ACR50 at 20 mg, 27% of patients reached ACR50 at 40 mg, and 20% of
patients reached ACR50 at 80 mg, and only 2% of patients receiving
placebo reached ACR50. These data illustrate that subcutaneous
D2E7, particularly at a dose of 40 mg/week, generates a good
response.
Example 3
Biweekly, Subcutaneous Administration of an Anti-TNF.alpha.
Antibody Biweekly, Subcutaneous Administration of D2E7
[0132] The clinical effects, safety, immunogenicity, and tolerance
of RA patients with partial responses to MTX following every other
week subcutaneous (s.c.) injections of placebo or D2E7 at several
dose levels for up to twenty-four weeks in conjunction with
continued MTX treatment was investigated.
Study Design
[0133] A placebo-controlled, double-blind, randomized, multi-center
study in patients with RA, who had insufficient efficacy or
tolerability to MTX was performed. During the course of the trial,
patients were continued on a stable dose of MTX with dose ranges
specified in the inclusion criteria described below.
[0134] This study consisted of two portions: 1) a "wash-out period"
of four weeks prior to the administration of the first dose
medication, during which time DMARDs (except for MTX) were
withdrawn; and 2) a "placebo controlled period" during which time
patients were randomized to one of four cohorts of sixty-seven
patients to receive placebo, 20, 40, or 80 mg D2E7 (as a total body
dose) given every other week s.c. for up to 24 weeks. Each dose of
study drug was administered as two s.c. injections of 1.6 mL each.
The patient's first dose was administered by medical personnel as
part of the patient's training. Subsequent doses were
self-administered by the patient at the study under the direct
observation of trained personnel for the first four weeks.
Thereafter, doses were administered outside the study site by the
patient, a trained individual designated by the patient, or by
medical personnel. Medication for four or five weeks was dispensed
after each clinical assessment. Patients were serially examined in
weeks one, two, three, four, six, eight, twelve, sixteen, twenty,
and twenty-four of the study with the joint examinations being
performed by a blinded assessor, independent of the treating
physician.
[0135] This study enrolled two hundred seventy-one patients with
RA. The study population was representative of the moderate to
severe RA population in North America: approximately 70% female,
and predominantly over the age of forty. The population was
selected using predetermined inclusion and exclusion criteria,
known to those of skill in the art e.g., a patient must have
received a diagnosis of RA as defined by the 1987-revised American
College of Rheumatology (ACR) criteria (set forth in Appendix
A)
Results
[0136] FIGS. 1B and 2-4 indicate that subcutaneous, biweekly D2E7
treatment combined with methotrexate was significantly better than
placebo in reducing the signs and symptoms of RA at twenty-four
weeks. All three doses of D2E7 were statistically significantly
more effective than placebo given weekly. Furthermore, D2E7 at 40
mg and 80 mg had better efficacy than the 20 mg dose.
EQUIVALENTS
[0137] 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 1
1
371107PRTArtificial Sequencemutated human antibody 1Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro
Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1052121PRTArtificial Sequencemutated human antibody 2Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12039PRTArtificial Sequencemutated human antibody 3Gln Arg Tyr Asn
Arg Ala Pro Tyr Xaa1 5412PRTArtificial Sequencemutated human
antibody 4Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5
1057PRTArtificial Sequencemutated human antibody 5Ala Ala Ser Thr
Leu Gln Ser1 5617PRTArtificial Sequencemutated human antibody 6Ala
Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu1 5 10
15Gly711PRTArtificial Sequencemutated human antibody 7Arg Ala Ser
Gln Gly Ile Arg Asn Tyr Leu Ala1 5 1085PRTArtificial
Sequencemutated human antibody 8Asp Tyr Ala Met His1
59107PRTArtificial Sequencemutated human antibody 9Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro
Tyr 85 90 95Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10510121PRTArtificial Sequencemutated human antibody 10Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40
45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60Glu Gly Arg Phe Ala Val Ser Arg Asp Asn Ala Lys Asn Ala Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Thr Lys Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu
Asp Asn Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120119PRTArtificial Sequencemutated human antibody 11Gln Lys Tyr
Asn Ser Ala Pro Tyr Ala1 5129PRTArtificial Sequencemutated human
antibody 12Gln Lys Tyr Asn Arg Ala Pro Tyr Ala1 5139PRTArtificial
Sequencemutated human antibody 13Gln Lys Tyr Gln Arg Ala Pro Tyr
Thr1 5149PRTArtificial Sequencemutated human antibody 14Gln Lys Tyr
Ser Ser Ala Pro Tyr Thr1 5159PRTArtificial Sequencemutated human
antibody 15Gln Lys Tyr Asn Ser Ala Pro Tyr Thr1 5169PRTArtificial
Sequencemutated human antibody 16Gln Lys Tyr Asn Arg Ala Pro Tyr
Thr1 5179PRTArtificial Sequencemutated human antibody 17Gln Lys Tyr
Asn Ser Ala Pro Tyr Tyr1 5189PRTArtificial Sequencemutated human
antibody 18Gln Lys Tyr Asn Ser Ala Pro Tyr Asn1 5199PRTArtificial
Sequencemutated human antibody 19Gln Lys Tyr Thr Ser Ala Pro Tyr
Thr1 5209PRTArtificial Sequencemutated human antibody 20Gln Lys Tyr
Asn Arg Ala Pro Tyr Asn1 5219PRTArtificial Sequencemutated human
antibody 21Gln Lys Tyr Asn Ser Ala Ala Tyr Ser1 5229PRTArtificial
Sequencemutated human antibody 22Gln Gln Tyr Asn Ser Ala Pro Asp
Thr1 5239PRTArtificial Sequencemutated human antibody 23Gln Lys Tyr
Asn Ser Asp Pro Tyr Thr1 5249PRTArtificial Sequencemutated human
antibody 24Gln Lys Tyr Ile Ser Ala Pro Tyr Thr1 5259PRTArtificial
Sequencemutated human antibody 25Gln Lys Tyr Asn Arg Pro Pro Tyr
Thr1 5269PRTArtificial Sequencemutated human antibody 26Gln Arg Tyr
Asn Arg Ala Pro Tyr Ala1 52712PRTArtificial Sequencemutated human
antibody 27Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn1 5
102812PRTArtificial Sequencemutated human antibody 28Ala Ser Tyr
Leu Ser Thr Ser Ser Ser Leu Asp Lys1 5 102912PRTArtificial
Sequencemutated human antibody 29Ala Ser Tyr Leu Ser Thr Ser Ser
Ser Leu Asp Tyr1 5 103012PRTArtificial Sequencemutated human
antibody 30Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp1 5
103112PRTArtificial Sequencemutated human antibody 31Ala Ser Tyr
Leu Ser Thr Ser Phe Ser Leu Asp Tyr1 5 103212PRTArtificial
Sequencemutated human antibody 32Ala Ser Tyr Leu Ser Thr Ser Ser
Ser Leu His Tyr1 5 103312PRTArtificial Sequencemutated human
antibody 33Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr1 5
103412PRTArtificial Sequencemutated human antibody 34Ala Ser Tyr
Leu Ser Thr Ala Ser Ser Leu Glu Tyr1 5 103512PRTArtificial
Sequencemutated human antibody 35Val Ser Tyr Leu Ser Thr Ala Ser
Ser Leu Asp Asn1 5 1036321DNAArtificial Sequencemutated human
antibody 36gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtagggga
cagagtcacc 60atcacttgtc gggcaagtca gggcatcaga aattacttag cctggtatca
gcaaaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccactt
tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctacagcct 240gaagatgttg caacttatta
ctgtcaaagg tataaccgtg caccgtatac ttttggccag 300gggaccaagg
tggaaatcaa a 32137363DNAArtificial Sequencemutated human antibody
37gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggcaggtc cctgagactc
60tcctgtgcgg cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct
120ccagggaagg gcctggaatg ggtctcagct atcacttgga atagtggtca
catagactat 180gcggactctg tggagggccg attcaccatc tccagagaca
acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agctgaggat
acggccgtat attactgtgc gaaagtctcg 300taccttagca ccgcgtcctc
ccttgactat tggggccaag gtaccctggt caccgtctcg 360agt 363
* * * * *