U.S. patent application number 13/965155 was filed with the patent office on 2013-12-12 for methods of treating disorders using human antibodies that bind human tnfalpha.
This patent application is currently assigned to ABBVIE BIOTECHNOLOGY LTD. The applicant listed for this patent is ABBVIE BIOTECHNOLOGY LTD. Invention is credited to Deborah J. Allen, Hendricus R.J.M. Hoogenboom, Zehra Kaymakcalan, Boris Labkovsky, John A. Mankovich, Brian T. McGuinness, Andrew J. Roberts, Paul Sakorafas, Jochen G. Salfeld, David Schoenhaut, Tristan J. Vaughan, Michael White, Alison J. Wilton.
Application Number | 20130330357 13/965155 |
Document ID | / |
Family ID | 26707297 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130330357 |
Kind Code |
A1 |
Salfeld; Jochen G. ; et
al. |
December 12, 2013 |
METHODS OF TREATING DISORDERS USING HUMAN ANTIBODIES THAT BIND
HUMAN TNFalpha
Abstract
Human antibodies, preferably recombinant human antibodies, that
specifically bind to human tumor necrosis factor .alpha.
(hTNF.alpha.) are disclosed. 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. The antibodies, or
antibody portions, of the invention are useful for detecting
hTNF.alpha. and for inhibiting hTNF.alpha. activity, e.g., in a
human subject suffering from a disorder in which hTNF.alpha.
activity is detrimental. Nucleic acids, vectors and host cells for
expressing the recombinant human antibodies of the invention, and
methods of synthesizing the recombinant human antibodies, are also
encompassed by the invention.
Inventors: |
Salfeld; Jochen G.; (North
Grafton, MA) ; Allen; Deborah J.; (London, GB)
; Kaymakcalan; Zehra; (Westborough, MA) ;
Labkovsky; Boris; (Marlborough, MA) ; Mankovich; John
A.; (Andover, MA) ; McGuinness; Brian T.;
(Cambridge, GB) ; Roberts; Andrew J.; (Cambridge,
GB) ; Sakorafas; Paul; (Newton Highlands, MA)
; Hoogenboom; Hendricus R.J.M.; (Hasselt, BE) ;
Schoenhaut; David; (Clifton, NJ) ; Vaughan; Tristan
J.; (Cambridge, GB) ; White; Michael;
(Framingham, MA) ; Wilton; Alison J.; (Cambridge,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE BIOTECHNOLOGY LTD |
Hamilton |
|
BM |
|
|
Assignee: |
ABBVIE BIOTECHNOLOGY LTD
Hamilton
BM
|
Family ID: |
26707297 |
Appl. No.: |
13/965155 |
Filed: |
August 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13736931 |
Jan 8, 2013 |
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13965155 |
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12578487 |
Oct 13, 2009 |
8372400 |
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|
13736931 |
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|
12369451 |
Feb 11, 2009 |
8206714 |
|
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12578487 |
|
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|
11787901 |
Apr 17, 2007 |
7541031 |
|
|
12369451 |
|
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09801185 |
Mar 7, 2001 |
7223394 |
|
|
11787901 |
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09125098 |
Mar 16, 1999 |
6258562 |
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PCT/US97/02219 |
Feb 10, 1997 |
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09801185 |
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08599226 |
Feb 9, 1996 |
6090382 |
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09125098 |
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60031476 |
Nov 25, 1996 |
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Current U.S.
Class: |
424/158.1 ;
530/389.2 |
Current CPC
Class: |
A61P 31/22 20180101;
A61P 9/10 20180101; A61P 7/02 20180101; A61P 39/02 20180101; A61P
3/04 20180101; A61P 31/18 20180101; A61P 31/20 20180101; C07K
16/241 20130101; A61P 27/02 20180101; A61P 19/08 20180101; A61K
2039/505 20130101; A61P 1/04 20180101; A61P 17/02 20180101; A61P
1/16 20180101; A61P 7/04 20180101; A61P 19/06 20180101; A61P 43/00
20180101; A61P 11/16 20180101; A61P 9/00 20180101; A61P 33/06
20180101; A61P 31/00 20180101; A61P 29/02 20180101; A61P 37/06
20180101; A61P 1/02 20180101; Y02A 50/30 20180101; C07K 2317/565
20130101; A61P 19/00 20180101; A61P 35/04 20180101; A61P 37/04
20180101; A61P 17/00 20180101; A61P 11/00 20180101; A61P 19/02
20180101; C07K 2317/56 20130101; A61P 21/00 20180101; A61P 37/02
20180101; A61P 3/10 20180101; A61P 13/12 20180101; A61K 38/00
20130101; A61P 1/00 20180101; A61P 7/00 20180101; A61P 31/04
20180101; A61P 9/04 20180101; A61P 35/00 20180101; A61P 37/00
20180101; A61P 37/08 20180101; A61P 25/00 20180101; A61P 29/00
20180101; Y10S 424/81 20130101; A61P 31/12 20180101; A61P 9/08
20180101; C07K 2317/21 20130101 |
Class at
Publication: |
424/158.1 ;
530/389.2 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. An isolated human antibody, or antigen-binding portion thereof,
comprising 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.
2. The antibody of claim 1, wherein the antigen-binding portion is
a fragment selected from the group consisting of an Fd fragment, a
Fab fragment, a F(ab')2, and a single chain Fv fragment.
3. The antibody of claim 1, which is an IgG.
4. The antibody of claim 1, which comprises an IgG1 heavy chain
constant region.
5. The antibody of claim 1, which comprises an IgG4 heavy chain
constant region.
6. A method of treating a subject having a disorder in which
TNF.alpha. is detrimental, comprising administering an isolated
human antibody, or an antigen-binding portion thereof, comprising 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, such that the
disorder in which TNF.alpha. is detrimental is treated.
7. The method of claim 6, wherein the disorder is an autoimmune
disease.
8. The method of claim 7, wherein the autoimmune disease is
selected from the group consisting of: rheumatoid arthritis,
rheumatoid spondylitis, osteoarthritis, gouty arthritis, an
allergy, multiple sclerosis, autoimmune diabetes, autoimmune
uveitis and nephrotic syndrome.
9. The method of claim 6, wherein the disorder is an infectious
disease.
10. The method of claim 6, wherein the disorder is transplant
rejection or graft-versus-host disease.
11. The method of claim 6, wherein the disorder is a
malignancy.
12. The method of claim 6, wherein the disorder is a pulmonary
disorder.
13. The method of claim 6, wherein the disorder is an intestinal
disorder.
14. The method of claim 13, wherein the intestinal disorder is
Crohn's disease or ulcerative colitis.
15. The method of claim 6, wherein the disorder is a cardiac
disorder.
16. The method of claim 6, wherein the disorder is selected from
the group consisting of inflammatory bone disorders, bone
resorption disease, alcoholic hepatitis, viral hepatitis,
coagulation disturbances, burns, reperfusion injury, keloid
formation, scar tissue formation and pyrexia.
17. The method of claim 6, wherein the antibody is an IgG.
18. The method of claim 6, wherein the antibody comprises an IgG1
heavy chain constant region.
19. The method of claim 6, wherein the antibody comprises an IgG4
heavy chain constant region.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/736,931, filed on Jan. 8, 2013, which is a continuation of U.S.
Ser. No. 12/578,487, filed on Oct. 13, 2009, now issued as U.S.
Pat. No. 8,372,400, which is a continuation of U.S. Ser. No.
12/369,451, filed on Feb. 11, 2009, now issued as U.S. Pat. No.
8,206,714, which is a continuation of U.S. Ser. No. 11/787,901,
filed on Apr. 17, 2007, now issued as U.S. Pat. No. 7,541,031,
which is a continuation application of U.S. Ser. No. 09/801,185,
filed on Mar. 7, 2001, now issued as U.S. Pat. No. 7,223,394, which
is a continuation of U.S. Ser. No. 09/125,098 filed on Mar. 16,
1999, now issued as U.S. Pat. No. 6,258,562, which claims priority
to International Application Serial No. PCT/US97/02219 filed Feb.
10, 1997, which claims priority to U.S. provisional Application
Ser. No. 60/031,476 filed Nov. 25, 1996. International Application
Serial No. PCT/US97/02219 is also a continuation-in-part of U.S.
application Ser. No. 08/599,226 filed Feb. 9, 1996. The contents of
each of the above applications and patents are expressly
incorporated by reference herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 9, 2013, is named 11781304319SeqList.txt and is 12,635
bytes in size.
BACKGROUND OF THE INVENTION
[0003] Tumor necrosis factor .alpha. (TNF.alpha.) is a cytokine
produced by numerous cell types, including monocytes and
macrophages, that was originally identified based on its capacity
to induce the necrosis of certain mouse tumors (see e.g., Old, L.
(1985) Science 230:630-632). Subsequently, a factor termed
cachectin, associated with cachexia, was shown to be the same
molecule as TNF.alpha.. TNF.alpha. has been implicated in mediating
shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev.
Biochem. 57:505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev.
Immunol. 7:625-655). Furthermore, TNF.alpha. has been implicated in
the pathophysiology of a variety of other human diseases and
disorders, including sepsis, infections, autoimmune diseases,
transplant rejection and graft-versus-host disease (see e.g.,
Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A., et al. Vasilli, P. (1992) Annu. Rev.
Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev.
Med. 45:491-503).
[0004] Because of the harmful role of human TNF.alpha.
(hTNF.alpha.) in a variety of human disorders, therapeutic
strategies have been designed to inhibit or counteract hTNF.alpha.
activity. In particular, antibodies that bind to, and neutralize,
hTNF.alpha. have been sought as a means to inhibit hTNF.alpha.
activity. Some of the earliest of such antibodies were mouse
monoclonal antibodies (mAbs), secreted by hybridomas prepared from
lymphocytes of mice immunized with hTNF.alpha. (see e.g., Hahn T;
et al., (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, C-M.,
et al. (1986) Biochem. Biophys. Res. Commun. 137:847-854; Hirai,
M., et al. (1987) J. Immunol. Methods 96:57-62; Fendly, B. M., et
al. (1987) Hybridoma 6:359-370; Moeller, A., et al. (1990) Cytokine
2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European
Patent Publication No. 186 833 B1 by Wallach, D.; European Patent
Application Publication No. 218 868 A1 by Old et al.; European
Patent Publication No. 260 610 B1 by Moeller, A., et al.). While
these mouse anti-hTNF.alpha. antibodies often displayed high
affinity for hTNF.alpha. (e.g., Kd.ltoreq.10.sup.-9M) and were able
to neutralize hTNF.alpha. activity, their use in vivo may be
limited by problems associated with administration of mouse
antibodies to humans, such as short serum half life, an inability
to trigger certain human effector functions and elicitation of an
unwanted immune response against the mouse antibody in a human (the
"human anti-mouse antibody" (HAMA) reaction).
[0005] 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).
[0006] 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).
[0007] 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.)
[0008] Accordingly, human antibodies, such as 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, are
still needed.
SUMMARY OF THE INVENTION
[0009] This invention provides human antibodies, 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. Antibodies of the
invention are further characterized by binding to hTNF.alpha. but
not hTNF.beta. (lymphotoxin) and by having the ability to bind to
other primate TNF.alpha.s and non-primate TNF.alpha.s in addition
to human TNF.alpha..
[0010] The antibodies of the invention 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 or scFv fragment). 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. 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.
[0011] In one embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, that dissociates
from human TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less
and a K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or
less, both determined by surface plasmon resonance, and neutralizes
human TNF.alpha. cytotoxicity in a standard in vitro L929 assay
with an IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably,
the isolated human antibody, or antigen-binding portion thereof,
dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less, or even more preferably, with a
K.sub.off of 1.times.10.sup.-4 s.sup.-1 or less. More preferably,
the isolated human antibody, or antigen-binding portion thereof,
neutralizes human TNF.alpha. cytotoxicity in a standard in vitro
L929 assay with an IC.sub.50 of 1.times.10.sup.-8 M or less, even
more preferably with an IC.sub.50 of 1.times.10.sup.-9 M or less
and still more preferably with an IC.sub.50 of 5.times.10.sup.-10 M
or less.
[0012] In another embodiment, the invention provides a human
antibody, or antigen-binding portion thereof, with the following
characteristics:
[0013] a) dissociates from human TNF.alpha. with a K.sub.off of
1.times.10.sup.-3 5.sup.-1 or less, as determined by surface
plasmon resonance;
[0014] 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;
[0015] 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.
[0016] 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 5.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.
[0017] In yet another embodiment, the invention provides a human
antibody, or an antigen-binding portion thereof, with 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.
[0018] In still another embodiment, the invention provides an
isolated human antibody, or an antigen binding portion thereof,
with 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.
[0019] In still other embodiments, the invention provides
antibodies, or antigen-binding portions thereof, with 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.
[0020] In yet another embodiment, the invention provides an
isolated human antibody, or antigen-binding portion thereof, that
neutralizes the activity of human TNF.alpha. but not human
TNF.beta. (lymphotoxin). In a preferred embodiment, the human
antibody, or antigen-binding portion thereof, neutralizes the
activity of human TNF.alpha., chimpanzee TNF.alpha. and at least
one additional primate TNF.alpha. selected from the group
consisting of baboon TNF.alpha., marmoset TNF.alpha., cynomolgus
TNF.alpha. and rhesus TNF.alpha.. Preferably, the antibody also
neutralizes the activity of at least one non-primate TNF.alpha..
For example, in one subembodiment, the isolated human antibody, or
antigen-binding portion thereof, also neutralizes the activity of
canine TNF.alpha.. In another subembodiment, the isolated human
antibody, or antigen-binding portion thereof, also neutralizes the
activity of pig TNF.alpha.. In yet another subembodiment, the
isolated human antibody, or antigen-binding portion thereof, also
neutralizes the activity of mouse TNF.alpha..
[0021] Another aspect of the invention pertains to nucleic acid
molecules encoding the antibodies, or antigen-binding portions, of
the invention. A preferred nucleic acid of the invention, encoding
a D2E7 LCVR, has the nucleotide sequence shown in FIG. 7 and SEQ ID
NO 36. Another preferred nucleic acid of the invention, encoding a
D2E7 HCVR, has the nucleotide sequence shown in FIG. 8 and SEQ ID
NO 37. Recombinant expression vectors carrying the
antibody-encoding nucleic acids of the invention, and host cells
into which such vectors have been introduced, are also encompassed
by the invention, as are methods of making the antibodies of the
invention by culturing the host cells of the invention.
[0022] Yet another aspect of the invention pertains to methods for
inhibiting human TNF.alpha. activity using an antibody, or
antigen-binding portion thereof, of the invention. In one
embodiment, the method comprises contacting human TNF.alpha. with
the antibody of the invention, or antigen-binding portion thereof,
such that human TNF.alpha. activity is inhibited. In another
embodiment, the method comprises administering an antibody of the
invention, or antigen-binding portion thereof, to a human subject
suffering from a disorder in which TNF.alpha. activity is
detrimental such that human TNF.alpha. activity in the human
subject is inhibited. 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A and 1B show the amino acid sequences of the light
chain variable region of D2E7 (D2E7 VL; also shown in SEQ ID NO:
1), alanine-scan mutants of D2E7 VL (LD2E7*.A1, LD2E7*.A3,
LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8), the light chain
variable region of the D2E7-related antibody 2SD4 (2SD4 VL; also
shown in SEQ ID NO: 9) and other D2E7-related light chain variable
regions (EP B12, VL10E4, VL100A9, VL100D2, VL10F4, LOE5, VLLOF9,
VLL0F10, VLLOG7, VLLOG9, VLLOH1, VLLOH10, VL1B7, VL1C1, VL1C7,
VL0.1F4, VL0.1H8, LOE7, LOE7.A and LOE7.T). FIG. 1A shows the FR1,
CDR1, FR2 and CDR2 domains. FIG. 1B shows the FR3, CDR3 and FR4
domains. The light chain CDR1 ("CDR L1"), CDR2 ("CDR L2") and CDR3
("CDR L3") domains are boxed.
[0024] FIGS. 2A and 2B show the amino acid sequences of the heavy
chain variable region of D2E7 (D2E7 VH; also shown in SEQ ID NO:
2), alanine-scan mutants of D2E7 VH (HD2E7*.A1, HD2E7*.A2,
HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8
and HD2E7*.A9), the heavy chain variable region of the D2E7-related
antibody 2SD4 (2SD4 VH; also shown in SEQ ID NO: 10) and other
D2E7-related heavy chain variable regions (VH1B11, VH1D8, VH1A11,
VH1B12, VH1-D2, VH1E4, VH1F6, VH1G1, 3C--H2, VH1-D2.N and
VH1-D2.Y). FIG. 2A shows the FR1, CDR1, FR2 and CDR2 domains. FIG.
2B shows the FR3, CDR3 and FR4 domains. The heavy chain CDR1 ("CDR
H1"), CDR2 ("CDR H2") and CDR3 ("CDR H3") domains are boxed.
[0025] FIG. 3 is a graph depicting the inhibition of
TNF.alpha.-induced L929 cytotoxicity by the human anti-hTNF.alpha.
antibody D2E7, as compared to the murine anti-hTNF.alpha. antibody
MAK 195.
[0026] FIG. 4 is a graph depicting the inhibition of rhTNF.alpha.
binding to hTNF.alpha. receptors on U-937 cells by the human
anti-hTNF.alpha. antibody D2E7, as compared to the murine
anti-hTNF.alpha. antibody MAK 195.
[0027] FIG. 5 is a graph depicting the inhibition of
TNF.alpha.-induced ELAM-1 expression on HUVEC by the human
anti-hTNF.alpha. antibody D2E7, as compared to the murine
anti-hTNF.alpha. antibody MAK 195.
[0028] FIG. 6 is a bar graph depicting protection from
TNF.alpha.-induced lethality in D-galactosamine-sensitized mice by
administration of the human anti-hTNF.alpha. antibody D2E7 (black
bars), as compared to the murine anti-hTNF.alpha. antibody MAK 195
(hatched bars).
[0029] FIG. 7 shows the nucleotide sequence of the light chain
variable region of D2E7, with the predicted amino acid sequence
below the nucleotide sequence. The CDR L1, CDR L2 and CDR L3
regions are underlined.
[0030] FIG. 8 shows the nucleotide sequence of the heavy chain
variable region of D2E7, with the predicted amino acid sequence
below the nucleotide sequence. The CDR H1, CDR H2 and CDR H3
regions are underlined.
[0031] FIG. 9 is a graph depicting the effect of D2E7 antibody
treatment on the mean joint size of Tg197 transgenic mice as a
polyarthritis model.
DETAILED DESCRIPTION OF THE INVENTION
[0032] This invention pertains to 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.
Various aspects of the invention relate to antibodies and antibody
fragments, and pharmaceutical compositions thereof, as well as
nucleic acids, recombinant expression vectors and host cells for
making such antibodies and fragments. Methods of using the
antibodies of the invention to detect human TNF.alpha. or to
inhibit human TNF.alpha. activity, either in vitro or in vivo, are
also encompassed by the invention.
[0033] In order that the present invention may be more readily
understood, certain terms are first defined.
[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 hTNF.alpha. is described further in, for example,
Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al.
(1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989)
Nature 338:225-228. The term human TNF.alpha. is intended to
include recombinant human TNF.alpha. (rhTNF.alpha.), which can be
prepared by standard recombinant expression methods or purchased
commercially (R & D Systems, Catalog No. 210-TA, Minneapolis,
Minn.).
[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 VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. Other
forms of single chain antibodies, such as diabodies are also
encompassed. Diabodies are bivalent, bispecific antibodies in which
VH and VL domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)
Structure 2:1121-1123).
[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 TNF.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-TNF.alpha. antibody contains no other sequences
encoding other VH regions that bind antigens other than
TNF.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 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 and has VL and VH sequences as shown in FIG. 1A, 1B
and FIG. 2A, 2B, respectively (the amino acid sequence of the D2E7
VL region is also shown in SEQ ID NO: 1; the amino acid sequence of
the D2E7 VH region is also shown in SEQ ID NO: 2). The binding
properties of D2E7, as compared to the murine anti-hTNF.alpha. MAK
195 mAb that exhibits high affinity and slow dissociation kinetics
and another human anti-hTNF.alpha. antibody related in sequence to
D2E7, 2SD4, are summarized below:
TABLE-US-00001 K.sub.off k.sub.on K.sub.d Stoichi- Antibody
sec.sup.-1 M.sup.-1 sec.sup.-1 M ometry D2E7 IgG1 8.81 .times.
10.sup.-5 1.91 .times. 10.sup.5 6.09 .times. 10.sup.-10 1.2 2SD4
IgG4 8.4 .times. 10.sup.-3 4.20 .times. 10.sup.5 2.00 .times.
10.sup.-8 0.8 MAK 195 F(ab').sub.2 8.70 .times. 10.sup.-5 1.90
.times. 10.sup.5 4.60 .times. 10.sup.-10 1.4
[0051] The D2E7 antibody, and related antibodies, also exhibit a
strong capacity to neutralize hTNF.alpha. activity, as assessed by
several in vitro and in vivo assays (see Example 4). For example,
these antibodies neutralize hTNF.alpha.-induced cytotoxicity of
L929 cells with IC.sub.50 values in the range of about 10.sup.-7 M
to about 10.sup.-10 M. D2E7, when expressed as a full-length IgG1
antibody, neutralizes hTNF.alpha.-induced cytotoxicity of L929
cells with IC.sub.50 of about 1.25.times.10.sup.-10 M. Moreover,
the neutralizing capacity of D2E7 is maintained when the antibody
is expressed as a Fab, F(ab').sub.2 or scFv fragment. D2E7 also
inhibits TNF.alpha.-induced cellular activation, as measured by
hTNF.alpha.-induced ELAM-1 expression on HUVEC (IC.sub.50=about
1.85.times.10.sup.-10 M), and binding of hTNF.alpha. to hTNF.alpha.
receptors on U-937 cells (IC.sub.50=about 1.56.times.10.sup.-10 M).
Regarding the latter, D2E7 inhibits the binding of hTNF.alpha. to
both the p55 and p75 hTNF.alpha. receptors. Furthermore, the
antibody inhibits hTNF.alpha.-induced lethality in vivo in mice
(ED.sub.50=1-2.5 .mu.g/mouse).
[0052] Regarding the binding specificity of D2E7, this antibody
binds to human TNF.alpha. in various forms, including soluble
hTNF.alpha., transmembrane hTNF.alpha. and hTNF.alpha. bound to
cellular receptors. D2E7 does not specifically bind to other
cytokines, such as lymphotoxin (TNF.beta.), IL-1.alpha.,
IL-1.beta., IL-2, IL-4, IL-6, IL-8, IFN.gamma. and TGF.beta..
However, D2E7 does exhibit crossreactivity to tumor necrosis
factors from other species. For example, the antibody neutralizes
the activity of at least five primate TNF.alpha.s (chimpanzee,
baboon, marmoset, cynomolgus and rhesus) with approximately
equivalent IC.sub.50 values as for neutralization of hTNF.alpha.
(see Example 4, subsection E). D2E7 also neutralizes the activity
of mouse TNF.alpha., although approximately 1000-fold less well
than human TNF.alpha. (see Example 4, subsection E). D2E7 also
binds to canine and porcine TNF.alpha..
[0053] In one aspect, the invention pertains to 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 an isolated human antibody,
or an antigen-binding portion thereof, that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably, the
isolated human antibody, or antigen-binding portion thereof,
dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less, or even more preferably, with a
K.sub.off of 1.times.10.sup.-4 s.sup.-1 or less. More preferably,
the isolated human antibody, or antigen-binding portion thereof,
neutralizes human TNF.alpha. cytotoxicity in a standard in vitro
L929 assay with an IC.sub.50 of 1.times.10.sup.-8 M or less, even
more preferably with an IC.sub.50 of 1.times.10.sup.-9 M or less
and still more preferably with an IC.sub.50 of 5.times.10.sup.-10 M
or less. In a preferred embodiment, the antibody is an isolated
human recombinant antibody, or an antigen-binding portion thereof.
In another preferred embodiment, the antibody also neutralizes
TNF.alpha.-induced cellular activation, as assessed using a
standard in vitro assay for TNF.alpha.-induced ELAM-1 expression on
human umbilical vein endothelial cells (HUVEC).
[0054] Surface plasmon resonance analysis for determining K.sub.d
and K.sub.off can be performed as described in Example 1. A
standard in vitro L929 assay for determining IC.sub.50 values is
described in Example 4, subsection A. A standard in vitro assay for
TNF.alpha.-induced ELAM-1 expression on human umbilical vein
endothelial cells (HUVEC) is described in Example 4, subsection C.
Examples of recombinant human antibodies that meet, or are
predicted to meet, the aforementioned kinetic and neutralization
criteria include antibodies having the following [VH/VL] pairs, the
sequences of which are shown in FIGS. 1A, 1B, 2A and 2B (see also
Examples 2, 3 and 4 for kinetic and neutralization analyses): [D2E7
VH/D2E7 VL]; [HD2E7*.A1/D2E7 VL], [HD2E7*.A2/D2E7 VL],
[HD2E7*.A3/D2E7 VL], [HD2E7*.A4/D2E7 VL], [HD2E7*.A5/D2E7 VL],
[HD2E7*.A6/D2E7 VL], [HD2E7*.A7/D2E7 VL], [HD2E7*.A8/D2E7 VL],
[HD2E7*.A9/D2E7 VL], [D2E7 VH/LD2E7*.A1], [D2E7 VH/LD2E7*.A4],
[D2E7 VH/LD2E7*.A5], [D2E7 VH/LD2E7*.A7], [D2E7 VH/LD2E7*.A8],
[HD2E7*.A9/LD2E7*.A1], [VH1-D2/LOE7], [VH1-D2.N/LOE7.1],
[VH1-D2.Y/LOE7.A], [VH1-D2.N/LOE7.A], [VH1-D2/EP B12] and
[3C--H2/LOE7].
[0055] 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 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. As demonstrated in
Example 3, 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 VH 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.. As shown in
Example 3, 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).
[0056] Accordingly, in another embodiment, the invention provides
an isolated human antibody, or antigen-binding portion thereof,
with the following characteristics:
[0057] 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;
[0058] 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;
[0059] 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.
[0060] 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.
[0061] In yet another embodiment, the invention provides an
isolated human antibody, or an antigen-binding portion thereof,
with 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. 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.
[0062] In still another embodiment, the invention provides an
isolated human antibody, or an antigen binding portion thereof,
with 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.
[0063] In still other embodiments, the invention provides an
isolated human antibody, or an antigen-binding portions thereof,
having 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.
[0064] In yet another embodiment, the invention provides a
recombinant human antibody, or antigen-binding portion thereof,
that neutralizes the activity of human TNF.alpha. but not human
TNF.beta.. Preferably, antibody, or antigen-binding portion
thereof, also neutralizes the activity of chimpanzee TNF.alpha. and
at least one additional primate TNF.alpha. selected from the group
consisting of baboon TNF.alpha., marmoset TNF.alpha., cynomolgus
TNF.alpha. and rhesus TNF.alpha.. Preferably, the antibody, or
antigen-binding portion thereof, neutralizes the human, chimpanzee
and/or additional primate TNF.alpha. in a standard in vitro L929
assay with an IC.sub.50 of 1.times.10.sup.-8 M or less, more
preferably 1.times.10.sup.-9 M or less, and even more preferably
5.times.10.sup.-10 M or less. In one subembodiment, the antibody
also neutralizes the activity of canine TNF.alpha., preferably in a
standard in vitro L929 assay with an IC.sub.50 of 1.times.10.sup.-7
M or less, more preferably 1.times.10.sup.-8 M or less and even
more preferably 5.times.10.sup.-9 M or less. In another
subembodiment, the antibody also neutralizes the activity of pig
TNF.alpha., preferably with an IC.sub.50 of 1.times.10.sup.-5 M or
less, more preferably 1.times.10.sup.-6 M or less and even more
preferably 5.times.10.sup.-7 M or less. In yet another embodiment,
the antibody also neutralizes the activity of mouse TNF.alpha.,
preferably with an IC.sub.50 of 1.times.10.sup.-4 M or less, more
preferably 1.times.10.sup.-5 M or less and even more preferably
5.times.10.sup.-6 M or less.
[0065] 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).
[0066] 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.
[0067] 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
[0068] 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 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.
[0069] 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.K 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.KI 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.
[0070] 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.
[0071] Moreover, it should be noted that if the "germline"
sequences obtained by PCR amplification encode amino acid
differences in the framework regions from the true germline
configuration (i.e., differences in the amplified sequence as
compared to the true germline sequence, for example as a result of
somatic mutation), it may be desirable to change these amino acid
differences back to the true germline sequences (i.e.,
"backmutation" of framework residues to the germline
configuration).
[0072] 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.
[0073] 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 (CH1, CH2 and CH3). 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.
[0074] 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.
[0075] 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).
[0076] 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).
[0077] 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.
[0078] 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).
[0079] 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).
[0080] 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), NS0 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.
[0081] 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.
[0082] 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 enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) 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.
[0083] In view of the foregoing, another aspect of the invention
pertains to nucleic acid, vector and host cell compositions that
can be used for recombinant expression of the antibodies and
antibody portions of the invention. The nucleotide sequence
encoding the D2E7 light chain variable region is shown in FIG. 7
and SEQ ID NO: 36. The CDR1 domain of the LCVR encompasses
nucleotides 70-102, the CDR2 domain encompasses nucleotides 148-168
and the CDR3 domain encompasses nucleotides 265-291. The nucleotide
sequence encoding the D2E7 heavy chain variable region is shown in
FIG. 8 and SEQ ID NO: 37. The CDR1 domain of the HCVR encompasses
nucleotides 91-105, the CDR2 domain encompasses nucleotides 148-198
and the CDR3 domain encompasses nucleotides 295-330. It will be
appreciated by the skilled artisan that nucleotide sequences
encoding D2E7-related antibodies, or portions thereof (e.g., a CDR
domain, such as a CDR3 domain), can be derived from the nucleotide
sequences encoding the D2E7 LCVR and HCVR using the genetic code
and standard molecular biology techniques.
[0084] In one embodiment, the invention provides an isolated
nucleic acid encoding a light chain CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 3 (i.e., the D2E7 VL CDR3), 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. This nucleic
acid can encode only the CDR3 region or, more preferably, encodes
an entire antibody light chain variable region (LCVR). For example,
the nucleic acid can encode an LCVR having a CDR2 domain comprising
the amino acid sequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2)
and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:
7 (i.e., the D2E7 VL CDR1).
[0085] In another embodiment, the invention provides an isolated
nucleic acid encoding a heavy chain CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 4 (i.e., the D2E7 VH CDR3), 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. This nucleic acid can encode only the CDR3
region or, more preferably, encodes an entire antibody heavy chain
variable region (HCVR). For example, the nucleic acid can encode a
HCVR having a CDR2 domain comprising the amino acid sequence of SEQ
ID NO: 6 (i.e., the D2E7 VH CDR2) and a CDR1 domain comprising the
amino acid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1).
[0086] In yet another embodiment, the invention provides isolated
nucleic acids encoding a D2E7-related CDR3 domain, e.g., comprising
an amino acid sequence selected from the group consisting of: SEQ
ID NO: 3, SEQ ID NO 4, 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, 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.
[0087] In still another embodiment, the invention provides an
isolated nucleic acid encoding an antibody light chain variable
region comprising the amino acid sequence of SEQ ID NO: 1 (i.e.,
the D2E7 LCVR). Preferably this nucleic acid comprises the
nucleotide sequence of SEQ ID NO: 36, although the skilled artisan
will appreciate that due to the degeneracy of the genetic code,
other nucleotide sequences can encode the amino acid sequence of
SEQ ID NO: 1. The nucleic acid can encode only the LCVR or can also
encode an antibody light chain constant region, operatively linked
to the LCVR. In one embodiment, this nucleic acid is in a
recombinant expression vector.
[0088] In still another embodiment, the invention provides an
isolated nucleic acid encoding an antibody heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 2 (i.e.,
the D2E7 HCVR). Preferably this nucleic acid comprises the
nucleotide sequence of SEQ ID NO: 37, although the skilled artisan
will appreciate that due to the degeneracy of the genetic code,
other nucleotide sequences can encode the amino acid sequence of
SEQ ID NO: 2. The nucleic acid can encode only the HCVR or can also
encode a heavy chain constant region, operatively linked to the
HCVR. For example, the nucleic acid can comprise an IgG1 or IgG4
constant region. In one embodiment, this nucleic acid is in a
recombinant expression vector.
[0089] The invention also provides recombinant expression vectors
encoding both an antibody heavy chain and an antibody light chain.
For example, in one embodiment, the invention provides a
recombinant expression vector encoding:
[0090] a) an antibody light chain having a variable region
comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the D2E7
LCVR); and
[0091] b) an antibody heavy chain having a variable region
comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the D2E7
HCVR).
[0092] The invention also provides host cells into which one or
more of the recombinant expression vectors of the invention have
been introduced. Preferably, the host cell is a mammalian host
cell, more preferably the host cell is a CHO cell, an NS0 cell or a
COS cell.
[0093] Still further the invention provides a method of
synthesizing a recombinant human antibody of the invention by
culturing a host cell of the invention in a suitable culture medium
until a recombinant human antibody of the invention is synthesized.
The method can further comprise isolating the recombinant human
antibody from the culture medium.
III. Selection of Recombinant Human Antibodies
[0094] Recombinant human antibodies of the invention in addition to
the D2E7 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 SurfZAP.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; Garrad 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.
[0095] 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, or guided
selection, 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.
[0096] 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.
[0097] The amino acid sequences of selected antibody heavy and
light chains can be compared to germline heavy and light chain
amino acid sequences. In cases where certain framework residues of
the selected VL and/or VH chains differ from the germline
configuration (e.g., as a result of somatic mutation of the
immunoglobulin genes used to prepare the phage library), it may be
desirable to "backmutate" the altered framework residues of the
selected antibodies to the germline configuration (i.e., change the
framework amino acid sequences of the selected antibodies so that
they are the same as the germline framework amino acid sequences).
Such "backmutation" (or "germlining") of framework residues can be
accomplished by standard molecular biology methods for introducing
specific mutations (e.g., site-directed mutagenesis; PCR-mediated
mutagenesis, and the like).
[0098] 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
[0099] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention 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. 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.
[0100] 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 or subcutaneous injection.
[0101] 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.
[0102] 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 intravenous injection or infusion.
As will be appreciated by the skilled artisan, the route and/or
mode of administration will vary depending upon the desired
results. 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, 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.
[0103] 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.
[0104] 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 that are useful for
treating disorders in which TNF.alpha. activity is detrimental. For
example, an anti-hTNF.alpha. antibody or antibody portion of the
invention may be coformulated and/or coadministered with 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.
[0105] Nonlimiting 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, S295; 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), S284; 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), 5282) and thalidomide-related drugs (e.g.,
Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see
e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), 5131; 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 (inhibitors 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.
[0106] Nonlimiting 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.
[0107] 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).
[0108] 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., 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 (CRAB) HP228 and
HP466 (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low
molecular peptide; SmithKline Beecham); tetravalent guanylhydrazone
CNI-1493 (Picower Institute); Tissue Factor Pathway Inhibitor
(TFPI; 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);
[0109] 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).
[0110] The use of the antibodies, or antibody portions, of the
invention in combination with other therapeutic agents is discussed
further in subsection IV.
[0111] 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.
[0112] 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.
[0113] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.1-20 mg/kg, more preferably 1-10
mg/kg. 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.
IV. Uses of the Antibodies of the Invention
[0114] 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.
[0115] 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 rhTNF.alpha. standard bound to the
anti-hTNF.alpha. antibody.
[0116] 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 (discussed further in Example 4, subsection
E).
[0117] The antibodies and antibody portions of the invention are
capable of neutralizing hTNF.alpha. activity both in vitro and in
vivo (see Example 4). Moreover, at least some of the antibodies of
the invention, such as D2E7, can neutralize TNF.alpha. activity
from other species. Accordingly, the antibodies and antibody
portions of the invention can be used to inhibit TNF.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 hTNF.alpha., an antibody or antibody portion of the
invention can be added to the culture medium to inhibit hTNF.alpha.
activity in the culture.
[0118] In another embodiment, the invention provides a method for
inhibiting TNF.alpha. activity in a subject suffering from a
disorder in which TNF.alpha. activity is detrimental. TNF.alpha.
has been implicated in the pathophysiology of a wide variety of
disorders (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169;
U.S. Pat. No. 5,231,024 to Moeller et al.; European Patent
Publication No. 260 610 B1 by Moeller, A.). The invention provides
methods for TNF.alpha. activity in a subject suffering from such a
disorder, which method comprises administering to the subject an
antibody or antibody portion of the invention such that TNF.alpha.
activity in the subject is inhibited. Preferably, the TNF.alpha. is
human TNF.alpha. and the subject is a human subject. Alternatively,
the subject can be a mammal expressing a TNF.alpha. with which an
antibody of the invention cross-reacts. Still further the subject
can be a mammal into which has been introduced hTNF.alpha. (e.g.,
by administration of hTNF.alpha. or by expression of an hTNF.alpha.
transgene). 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).
[0119] As used herein, the term "a disorder in which TNF.alpha.
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF.alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF.alpha. activity is detrimental
is a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above. 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:
[0120] A. Sepsis
[0121] 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.,
Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A.; 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.
[0122] 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). Other combination
therapies for the treatment of sepsis are discussed further in
subsection III.
[0123] 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.).
[0124] B. Autoimmune Diseases
[0125] 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., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat.
No. 5,231,024 to Moeller et al.; European Patent Publication No.
260 610 B1 by Moeller, A.; Tracey and Cerami, supra; Arend, W. P.
and Dayer, J-M. (1995) Arth. Rheum. 38:151-160; Fava, R. A., et al.
(1993) Clin. Exp. Immunol. 94:261-266). TNF.alpha. also has been
implicated in promoting the death of islet cells and in mediating
insulin resistance in diabetes (see e.g., Tracey and Cerami, supra;
PCT Publication No. WO 94/08609). TNF.alpha. also has been
implicated in mediating cytotoxicity to oligodendrocytes and
induction of inflammatory plaques in multiple sclerosis (see e.g.,
Tracey and Cerami, supra). Chimeric and humanized murine
anti-hTNF.alpha. antibodies have undergone clinical testing for
treatment of rheumatoid arthritis (see e.g., Elliott, M. J., et al.
(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet
344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.
34:334-342).
[0126] 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). An antibody, or antibody portion, of the invention
also can be administered with one or more additional therapeutic
agents useful in the treatment of autoimmune diseases, as discussed
further in subsection III.
[0127] C. Infectious Diseases
[0128] 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.
TNF.alpha. also has been implicated in mediating brain
inflammation, inducing breakdown of the blood-brain bather,
triggering septic shock syndrome and activating venous infarction
in meningitis. TNF.alpha. also has been implicated in inducing
cachexia, stimulating viral proliferation and mediating central
nervous system injury in acquired immune deficiency syndrome
(AIDS). 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).
[0129] D. Transplantation
[0130] 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., 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.
[0131] E. Malignancy
[0132] Tumor necrosis factor has been implicated in inducing
cachexia, stimulating tumor growth, enhancing metastatic potential
and mediating cytotoxicity in malignancies. 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.
[0133] F. Pulmonary Disorders
[0134] Tumor necrosis factor has been implicated in the
pathophysiology of adult respiratory distress syndrome (ARDS),
including stimulating leukocyte-endothelial activation, directing
cytotoxicity to pneumocytes and inducing vascular leakage syndrome.
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. An antibody, or antibody portion, of the invention also
can be administered with one or more additional therapeutic agents
useful in the treatment of pulmonary disorders, as discussed
further in subsection III.
[0135] G. Intestinal Disorders
[0136] 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. An antibody, or
antibody portion, of the invention also can be administered with
one or more additional therapeutic agents useful in the treatment
of intestinal disorders, as discussed further in subsection
III.
[0137] H. Cardiac Disorders
[0138] 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).
[0139] I. Others
[0140] 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 Stern, P. H. (1993) Bone
14:871-876), hepatitis, including alcoholic hepatitis (see e.g.,
McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351;
Felver, M. E., et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259;
and Hansen, J., et al. (1994) Hepatology 20:461-474), 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), and
fulminant hepatitis; coagulation disturbances (see e.g., van der
Poll, T., et al. (1990) N. Engl. J. Med. 322:1622-1627; and van der
Poll, T., et al. (1991) Prog. Clin. Biol. Res. 367:55-60), burns
(see e.g., Giroir, B. P., et al. (1994) Am. J. Physiol.
267: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. J.
Physiol. 267:G1122-1127; Serrick, C., et al. (1994) Transplantation
58:1158-1162; and Yao, Y. M., et al. (1995) Resuscitation
29:157-168), keloid formation (see e.g., McCauley, R. L., et al.
(1992) J. Clin. Immunol. 12:300-308), scar tissue formation;
pyrexia; periodontal disease; obesity and radiation toxicity.
[0141] 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
Kinetic Analysis of Binding of Human Antibodies to hTNF.alpha.
[0142] Real-time binding interactions between ligand (biotinylated
recombinant human TNF.alpha. (rhTNF.alpha.) immobilized on a
biosensor matrix) and analyte (antibodies in solution) were
measured by surface plasmon resonance (SPR) using the BIAcore
system (Pharmacia Biosensor, Piscataway, N.J.). The system utilizes
the optical properties of SPR to detect alterations in protein
concentrations within a dextran biosensor matrix. Proteins are
covalently bound to the dextran matrix at known concentrations.
Antibodies are injected through the dextran matrix and specific
binding between injected antibodies and immobilized ligand results
in an increased matrix protein concentration and resultant change
in the SPR signal. These changes in SPR signal are recorded as
resonance units (RU) and are displayed with respect to time along
the y-axis of a sensorgram.
[0143] To facilitate immobilization of biotinylated rhTNF.alpha. on
the biosensor matrix, streptavidin is covalently linked via free
amine groups to the dextran matrix by first activating carboxyl
groups on the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400
mM N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride
(EDC). Next, streptavidin is injected across the activated matrix.
Thirty-five microliters of streptavidin (25 .mu.g/ml), diluted in
sodium acetate, pH 4.5, is injected across the activated biosensor
and free amines on the protein are bound directly to the activated
carboxyl groups. Unreacted matrix EDC-esters are deactivated by an
injection of 1 M ethanolamine Streptavidin-coupled biosensor chips
also are commercially available (Pharmacia BR-1000-16, Pharmacia
Biosensor, Piscataway, N.J.).
[0144] Biotinylated rhTNF.alpha. was prepared by first dissolving
5.0 mg of biotin (D-biotinyl-.epsilon.-aminocaproic acid
N-hydroxysuccinimide ester; Boehringer Mannheim Cat. No. 1008 960)
in 500 .mu.l dimethylsulfoxide to make a 10 mg/ml solution. Ten
microliters of biotin was added per ml of rhTNF.alpha. (at 2.65
mg/ml) for a 2:1 molar ratio of biotin to rhTNF.alpha.. The
reaction was mixed gently and incubated for two hours at room
temperature in the dark. A PD-10 column, Sephadex G-25M (Pharmacia
Catalog No. 17-0851-01) was equilibrated with 25 ml of cold PBS and
loaded with 2 ml of rhTNF.alpha.-biotin per column. The column was
eluted with 10.times.1 ml cold PBS. Fractions were collected and
read at OD280 (1.0 OD=1.25 mg/ml). The appropriate fractions were
pooled and stored at -80.degree. C. until use. Biotinylated
rhTNF.alpha. also is commercially available (R & D Systems
Catalog No. FTA00, Minneapolis, Minn.).
[0145] Biotinylated rhTNF.alpha. to be immobilized on the matrix
via streptavidin was diluted in PBS running buffer (Gibco Cat. No.
14190-144, Gibco BRL, Grand Island, N.Y.) supplemented with 0.05%
(BIAcore) surfactant P20 (Pharmacia BR-1000-54, Pharmacia
Biosensor, Piscataway, N.J.). To determine the capacity of
rhTNF.alpha.-specific antibodies to bind immobilized rhTNF.alpha.,
a binding assay was conducted as follows. Aliquots of biotinylated
rhTNF.alpha. (25 nM; 10 .mu.l aliquots) were injected through the
streptavidin-coupled dextran matrix at a flow rate of 5 .mu.l/min.
Before injection of the protein and immediately afterward, PBS
buffer alone flowed through each flow cell. The net difference in
signal between baseline and approximately 30 sec. after completion
of biotinylated rhTNF.alpha. injection was taken to represent the
binding value (approximately 500 RU). Direct rhTNF.alpha.-specific
antibody binding to immobilized biotinylated rhTNF.alpha. was
measured. Antibodies (20 .mu.g/ml) were diluted in PBS running
buffer and 25 .mu.l aliquots were injected through the immobilized
protein matrices at a flow rate of 5 .mu.l/min Prior to injection
of antibody, and immediately afterwards, PBS buffer alone flowed
through each flow cell. The net difference in baseline signal and
signal after completion of antibody injection was taken to
represent the binding value of the particular sample. Biosensor
matrices were regenerated using 100 mM HCl before injection of the
next sample. To determine the off rate (K.sub.off), on rate
(K.sub.on), association rate (K.sub.a) and dissociation rate
(K.sub.d) constants, BIAcore kinetic evaluation software (version
2.1) was used.
[0146] Representative results of D2E7 (IgG4 full-length antibody)
binding to biotinylated rhTNF.alpha., as compared to the mouse mAb
MAK 195 (F(ab').sub.2 fragment), are shown below in Table 1.
TABLE-US-00002 TABLE 1 Binding of D2E7 IgG4 or MAK 195 to
Biotinylated rhTNF.alpha. K.sub.off, [Ab], rhTNF.alpha., Ab, bound,
rhTNF.alpha./ sec.sup.-1, Antibody nM bound, RUs RUs Ab (Avg) D2E7
267 373 1215 1.14 8.45 .times. 10.sup.-5 133 420 1569 1.30 5.42
.times. 10.sup.-5 67 434 1633 1.31 4.75 .times. 10.sup.-5 33 450
1532 1.19 4.46 .times. 10.sup.-5 17 460 1296 0.98 3.47 .times.
10.sup.-5 8 486 936 0.67 2.63 .times. 10.sup.-5 4 489 536 0.38 2.17
.times. 10.sup.-5 2 470 244 0.18 3.68 .times. 10.sup.-5 (4.38
.times. 10.sup.-5) MAK 195 400 375 881 1.20 5.38 .times. 10.sup.-5
200 400 1080 1.38 4.54 .times. 10.sup.-5 100 419 1141 1.39 3.54
.times. 10.sup.-5 50 427 1106 1.32 3.67 .times. 10.sup.-5 25 446
957 1.09 4.41 .times. 10.sup.-5 13 464 708 0.78 3.66 .times.
10.sup.-5 6 474 433 0.47 7.37 .times. 10.sup.-5 3 451 231 0.26 6.95
.times. 10.sup.-5 (4.94 .times. 10.sup.-5)
[0147] In a second series of experiments, the molecular kinetic
interactions between an IgG1 full-length form of D2E7 and
biotinylated rhTNF was quantitatively analyzed using BIAcore
technology, as described above, and kinetic rate constants were
derived, summarized below in Tables 2, 3 and 4.
TABLE-US-00003 TABLE 2 Apparent dissociation rate constants of the
interaction between D2E7 and biotinylated rhTNF Experiment K.sub.d
(s.sup.-1) 1 9.58 .times. 10.sup.-5 2 9.26 .times. 10.sup.-5 3 7.60
.times. 10.sup.-5 Average 8.81 .+-. 1.06 .times. 10.sup.-5
TABLE-US-00004 TABLE 3 Apparent association rate constants of the
interaction between D2E7 and biotinylated rhTNF Experiment K.sub.a
(M.sup.-1, s.sup.-1) 1 1.33 .times. 10.sup.5 2 1.05 .times.
10.sup.5 3 3.36 .times. 10.sup.5 Average 1.91 .+-. 1.26 .times.
10.sup.5
TABLE-US-00005 TABLE 4 Apparent kinetic reate and affinity
constants of D2E7 and biotinylated rhTNF Experiment K.sub.a
(M.sup.-1, s.sup.-1) K.sub.d (s.sup.-1) K.sub.d (M) 1 1.33 .times.
10.sup.5 9.58 .times. 10.sup.-5 7.20 .times. 10.sup.-10 2 1.05
.times. 10.sup.5 9.26 .times. 10.sup.-5 8.82 .times. 10.sup.-10 3
3.36 .times. 10.sup.5 7.60 .times. 10.sup.-5 2.26 .times.
10.sup.-10 Average 1.91 .+-. 8.81 .+-. 6.09 .+-. 1.26 .times.
10.sup.5 1.06 .times. 10.sup.-5 3.42 .times. 10.sup.-10
Dissociation and association rate constants were calculated by
analyzing the dissociation and association regions of the
sensorgrams by BIA analysis software. Conventional chemical
reaction kinetics were assumed for the interaction between D2E7 and
biotinylated rhTNF molecule: a zero order dissociation and first
order association kinetics. For the sake of analysis, interaction
only between one arm of the bivalent D2E7 antibody and one unit of
the trimeric biotinylated rhTNF was considered in choosing
molecular models for the analysis of the kinetic data. Three
independent experiments were performed and the results were
analyzed separately. The average apparent dissociation rate
constant (k.sub.d) of the interaction between D2E7 and biotinylated
rhTNF was 8.81.+-.1.06.times.10.sup.-5 s.sup.-1, and the average
apparent association rate constant, k.sub.a was
1.91.+-.1.26.times.10.sup.5 M.sup.-1 s.sup.-1. The apparent
intrinsic dissociation constant (K.sub.d) was then calculated by
the formula: K.sub.d=k.sub.d/k.sub.a. Thus, the mean K.sub.d of
D2E7 antibody for rhTNF derived from kinetic parameters was
6.09.+-.3.42.times.10.sup.-10 M. Minor differences in the kinetic
values for the IgG1 form of D2E7 (presented in Tables 2, 3 and 4)
and the IgG4 form of D2E7 (presented in Table 1 and in Examples 2
and 3) are not thought to be true differences resulting from the
presence of either an IgG1 or an IgG4 constant regions but rather
are thought to be attributable to more accurate antibody
concentration measurements used for the IgG1 kinetic analysis.
Accordingly, the kinetic values for the IgG1 form of D2E7 presented
herein are thought to be the most accurate kinetic parameters for
the D2E7 antibody.
Example 2
Alanine Scanning Mutagenesis of D2E7 CDR3Domains
[0148] A series of single alanine mutations were introduced by
standard methods along the CDR3 domain of the D2E7 VL and the D2E7
VH regions. The light chain mutations are illustrated in FIG. 1B
(LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and
LD2E7*.A8, having an alanine mutation at position 1, 3, 4, 5, 7 or
8, respectively, of the D2E7 VL CDR3 domain). The heavy chain
mutations are illustrated in FIG. 2B (HD2E7*.A1, HD2E7*.A2,
HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8
and HD2E7*.A9, having an alanine mutation at position 2, 3, 4, 5,
6, 8, 9, 10 or 11, respectively, of the D2E7 VH CDR3 domain). The
kinetics of rhTNF.alpha. interaction with an antibody composed of
wild-type D2E7 VL and VH was compared to that of antibodies
composed of 1) a wild-type D2E7 VL paired with an
alanine-substituted D2E7 VH; 2) a wild-type D2E7 VH paired with an
alanine-substituted D2E7 VL; or 3) an alanine-substituted D2E7 VL
paired with an alanine-substituted D2E7 VH. All antibodies were
tested as full-length, IgG4 molecules.
[0149] Kinetics of interaction of antibodies with rhTNF.alpha. was
determined by surface plasmon resonance as described in Example 1.
The K.sub.off rates for the different VH/VL pairs are summarized
below in Table 5:
TABLE-US-00006 TABLE 5 Binding of D2E7 Alanine-Scan Mutants to
Biotinylated rhTNF.alpha. VH VL K.sub.off (sec.sup.-1) D2E7 VH D2E7
VL 9.65 .times. 10.sup.-5 HD2E7*.A1 D2E7 VL 1.4 .times. 10.sup.-4
HD2E7*.A2 D2E7 VL 4.6 .times. 10.sup.-4 HD2E7*.A3 D2E7 VL 8.15
.times. 10.sup.-4 HD2E7*.A4 D2E7 VL 1.8 .times. 10.sup.-4 HD2E7*.A5
D2E7 VL 2.35 .times. 10.sup.-4 HD2E7*.A6 D2E7 VL 2.9 .times.
10.sup.-4 HD2E7*.A7 D2E7 VL 1.0 .times. 10.sup.-4 HD2E7*.A8 D2E7 VL
3.1 .times. 10.sup.-4 HD2E7*.A9 D2E7 VL 8.1 .times. 10.sup.-4 D2E7
VH LD2E7*.A1 6.6 .times. 10.sup.-5 D2E7 VH LD2E7*.A3 NOT DETECTABLE
D2E7 VH LD2E7*.A4 1.75 .times. 10.sup.-4 D2E7 VH LD2E7*.A5 1.8
.times. 10.sup.-4 D2E7 VH LD2E7*.A7 1.4 .times. 10.sup.-4 D2E7 VH
LD2E7*.A8 3.65 .times. 10.sup.-4 HD2E7*.A9 LD2E7*.A1 1.05 .times.
10.sup.-4
[0150] These results demonstrate that the majority of positions of
the CDR3 domains of the D2E7 VL region and VH region are amenable
to substitution with a single alanine residue. Substitution of a
single alanine at position 1, 4, 5, or 7 of the D2E7 VL CDR3 domain
or at position 2, 5, 6, 8, 9 or 10 of the D2E7 VH CDR3 domain does
not significantly affect the off rate of hTNF.alpha. binding as
compared to the wild-type parental D2E7 antibody. Substitution of
alanine at position 8 of the D2E7 VL CDR3 or at position 3 of the
D2E7 VH CDR3 gives a 4-fold faster K.sub.off and an alanine
substitution at position 4 or 11 of D2E7 VH CDR3 gives an 8-fold
faster K.sub.off, indicating that these positions are more critical
for binding to hTNF.alpha.. However, a single alanine substitution
at position 1, 4, 5, 7 or 8 of the D2E7 VL CDR3 domain or at
position 2, 3, 4, 5, 6, 8, 9, 10 or 11 of the D2E7 VH CDR3 domain
still results in an anti-hTNF.alpha. antibody having a K.sub.off of
1.times.10.sup.-3 sec.sup.-1 or less.
Example 3
Binding Analysis of D2E7-Related Antibodies
[0151] A series of antibodies related in sequence to D2E7 were
analyzed for their binding to rhTNF.alpha., as compared to D2E7, by
surface plasmon resonance as described in Example 1. The amino acid
sequences of the VL regions tested are shown in FIGS. 1A and 1B.
The amino acid sequences of the VH regions tested are shown in
FIGS. 2A and 2B. The K.sub.off rates for various VH/VL pairs (in
the indicated format, either as a full-length IgG1 or IgG4 antibody
or as a scFv) are summarized below in Table 6:
TABLE-US-00007 TABLE 6 Binding of D2E7-Related Antibodies to
Biotinylated rhTNF.alpha. VH VL Format K.sub.off (sec.sup.-1) D2E7
VH D2E7 VL IgG1/IgG4 9.65 .times. 10.sup.-5 VH1-D2 LOE7 IgG1/IgG4
7.7 .times. 10.sup.-5 VH1-D2 LOE7 scFv 4.6 .times. 10.sup.-4
VH1-D2.N LOE7.T IgG4 2.1 .times. 10.sup.-5 VH1-D2.Y LOE7.A IgG4 2.7
.times. 10.sup.-5 VH1-D2.N LOE7.A IgG4 3.2 .times. 10.sup.-5 VH1-D2
EP B12 scFv 8.0 .times. 10.sup.-4 VH1-D2 2SD4 VL scFv 1.94 .times.
10.sup.-3 3C-H2 LOE7 scFv 1.5 .times. 10.sup.-3 2SD4 VH LOE7 scFv
6.07 .times. 10.sup.-3 2SD4 VH 2SD4 VL scFv 1.37 .times. 10.sup.-2
VH1A11 2SD4 VL scFv 1.34 .times. 10.sup.-2 VH1B12 2SD4 VL scFv 1.01
.times. 10.sup.-2 VH1B11 2SD4 VL scFv 9.8 .times. 10.sup.-3 VH1E4
2SD4 VL scFv 1.59 .times. 10.sup.-2 VH1F6 2SD4 VL scFv 2.29 .times.
10.sup.-2 VH1D8 2SD4 VL scFv 9.5 .times. 10.sup.-3 VH1G1 2SD4 VL
scFv 2.14 .times. 10.sup.-2 2SD4 VH EP B12 scFv 6.7 .times.
10.sup.-3 2SD4 VH VL10E4 scFv 9.6 .times. 10.sup.-3 2SD4 VH VL100A9
scFv 1.33 .times. 10.sup.-2 2SD4 VH VL100D2 scFv 1.41 .times.
10.sup.-2 2SD4 VH VL10F4 scFv 1.11 .times. 10.sup.-2 2SD4 VH VLLOE5
scFv 1.16 .times. 10.sup.-2 2SD4 VH VLL0F9 scFv 6.09 .times.
10.sup.-3 2SD4 VH VLL0F10 scFv 1.34 .times. 10.sup.-2 2SD4 VH
VLLOG7 scFv 1.56 .times. 10.sup.-2 2SD4 VH VLLOG9 scFv 1.46 .times.
10.sup.-2 2SD4 VH VLLOH1 scFv 1.17 .times. 10.sup.-2 2SD4 VH
VLLOH10 scFv 1.12 .times. 10.sup.-2 2SD4 VH VL1B7 scFv 1.3 .times.
10.sup.-2 2SD4 VH VL1C1 scFv 1.36 .times. 10.sup.-2 2SD4 VH VL1C7
scFv 2.0 .times. 10.sup.-2 2SD4 VH VL0.1F4 scFv 1.76 .times.
10.sup.-2 2SD4 VH VL0.1H8 scFv 1.14 .times. 10.sup.-2
[0152] The slow off rates (i.e., K.sub.off.ltoreq.1.times.10.sup.-4
sec.sup.-1) for full-length antibodies (i.e., IgG format) having a
VL selected from D2E7, LOE7, LOE7.T and LOE7.A, which have either a
threonine or an alanine at position 9, indicate that position 9 of
the D2E7 VL CDR3 can be occupied by either of these two residues
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). Furthermore,
the slow off rates (i.e., K.sub.off.ltoreq.1.times.10.sup.-4
sec.sup.-1) for antibodies having a VH selected from D2E7, VH1-D2.N
and VH1-D2.Y, which have either a tyrosine or an asparagine at
position 12, indicate that position 12 of the D2E7 VH CDR3 can be
occupied by either of these two residues without substantially
affecting the K.sub.off. Accordingly, a consensus motif for the
D2E7 VH CDR3 comprises the amino acid sequence:
V--S--Y-L-S-T-A-S--S-L-D-(Y/N) (SEQ ID NO: 4).
[0153] The results shown in Table 6 demonstrate that, in scFv
format, antibodies containing the 2SD4 VL or VH CDR3 region exhibit
a faster K.sub.off (i.e., K.sub.off.gtoreq.1.times.10.sup.-3
sec.sup.-1) as compared to antibodies containing the D2E7 VL or VH
CDR3 region. Within the VL CDR3, 2SD4 differs from D2E7 at
positions 2, 5 and 9. As discussed above, however, position 9 may
be occupied by Ala (as in 2SD4) or Thr (as in D2E7) without
substantially affecting the K.sub.off. Thus, by comparison of 2SD4
and D2E7, positions 2 and 5 of the D2E7 VL CDR3, both arginines,
can be identified as being critical for the association of the
antibody with hTNF.alpha.. These residues could be directly
involved as contact residues in the antibody binding site or could
contribute critically to maintaining the scaffolding architecture
of the antibody molecule in this region. Regarding the importance
of position 2, replacement of Arg (in LOE7, which has the same VL
CDR3 as D2E7) with Lys (in EP B 12) accelerates the off rate by a
factor of two. Regarding the importance of position 5, replacement
of Arg (in D2E7) with Ala (in LD2E7*.A5), as described in Example
2, also accelerates the off rate two-fold. Furthermore, without
either Arg at positions 2 and 5 (in 2SD4), the off rate is
five-fold faster. However, it should be noted that although
position 5 is important for improved binding to hTNF.alpha., a
change at this position can be negated by changes at other
positions, as seen in VLLOE4, VLLOH1 or VL0.1H8.
[0154] Within the VH CDR3, 2SD4 differs from D2E7 at positions 1, 7
and 12. As discussed above, however, position 12 may be occupied by
Asn (as in 2SD4) or Tyr (as in D2E7) without substantially
affecting the K.sub.off. Thus, by comparison of 2SD4 and D2E7,
positions 1 and 7 of the D2E7 VH CDR3 can be identified as being
critical for binding to hTNF.alpha.. As discussed above, these
residues could be directly involved as contact residues in the
antibody binding site or could contribute critically to maintaining
the scaffolding architecture of the antibody molecule in this
region. Both positions are important for binding to hTNF.alpha.
since when the 3C--H2 VH CDR3 (which has a valine to alanine change
at position 1 with respect to the D2E7 VH CDR3) is used, the scFv
has a 3-fold faster off rate than when the D2E7 VH CDR3 is used but
this off rate is still four times slower than when the 2SD4 VH CDR3
is used (which has changes at both positions 1 and 7 with respect
to the D2E7 VH CDR3).
Example 4
Functional Activity of D2E7
[0155] To examine the functional activity of D2E7, the antibody was
used in several assays that measure the ability of the antibody to
inhibit hTNF.alpha. activity, either in vitro or in vivo.
A. Neutralization of TNF.alpha.-Induced Cytotoxicity in L929
Cells
[0156] Human recombinant TNF.alpha. (rhTNF.alpha.) causes cell
cytotoxicity to murine L929 cells after an incubation period of
18-24 hours. Human anti-hTNF.alpha. antibodies were evaluated in
L929 assays by coincubation of antibodies with rhTNF.alpha. and the
cells as follows. A 96-well microtiter plate containing 100 .mu.l
of anti-hTNF.alpha. Abs was serially diluted 1/3 down the plate in
duplicates using RPMI medium containing 10% fetal bovine serum
(FBS). Fifty microliters of rhTNF.alpha. was added for a final
concentration of 500 pg/ml in each sample well. The plates were
then incubated for 30 minutes at room temperature. Next, 50 .mu.l
of TNF.alpha.-sensitive L929 mouse fibroblasts cells were added for
a final concentration of 5.times.10.sup.4 cells per well, including
1 .mu.g/ml Actinomycin-D. Controls included medium plus cells and
rhTNF.alpha. plus cells. These controls, and a TNF.alpha. standard
curve, ranging from 2 ng/ml to 8.2 pg/ml, were used to determine
the quality of the assay and provide a window of neutralization.
The plates were then incubated overnight (18-24 hours) at
37.degree. C. in 5% CO.sub.2.
[0157] One hundred microliters of medium was removed from each well
and 50 .mu.l of 5 mg/ml
3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT;
commercially available from Sigma Chemical Co., St. Louis, Mo.) in
PBS was added. The plates were then incubated for 4 hours at
37.degree. C. Fifty microliters of 20% sodium dodecyl sulfate (SDS)
was then added to each well and the plates were incubated overnight
at 37.degree. C. The optical density at 570/630 nm was measured,
curves were plotted for each sample and IC.sub.50s were determined
by standard methods.
[0158] Representative results for human antibodies having various
VL and VH pairs, as compared to the murine MAK 195 mAb, are shown
in FIG. 3 and in Table 7 below.
TABLE-US-00008 TABLE 7 Neutralization of TNF.alpha.-Induced L929
Cytotoxicity VH VL Structure IC.sub.50, M D2E7 D2E7 scFv 1.1
.times. 10.sup.-10 D2E7 D2E7 IgG4 4.7 .times. 10.sup.-11 2SD4 2SD4
scFv/IgG1/IgG4 3.0 .times. 10.sup.-7 2SD4 LOE7 scFv 4.3 .times.
10.sup.-8 VH1-D2 2SD4 scFv 1.0 .times. 10.sup.-8 VH1-D2 LOE7
scFv/IgG1/IgG4 3.4 .times. 10.sup.-10 VH1.D2.Y LOE7.T IgG4 8.1
.times. 10.sup.-11 VH1-D2.N LOE7.T IgG4 1.3 .times. 10.sup.-10
VH1-D2.Y LOE7.A IgG4 2.8 .times. 10.sup.-11 VH1-D2.N LOE7.A IgG4
6.2 .times. 10.sup.-11 MAK 195 MAK 195 scFv 1.9 .times. 10.sup.-8
MAK 195 MAK195 F(ab').sub.2 6.2 .times. 10.sup.-11
The results in FIG. 3 and Table 7 demonstrate that the D2E7 human
anti-hTNF.alpha. antibody, and various D2E7-related antibodies,
neutralize TNF.alpha.-induced L929 cytotoxicity with a capacity
approximately equivalent to that of the murine anti-hTNF.alpha. mAb
MAK 195.
[0159] In another series of experiments, the ability of the IgG1
form of D2E7 to neutralize TNF.alpha.-induced L929 cytotoxicity was
examined as described above. The results from three independent
experiments, and the average thereof, are summarized below in Table
8:
TABLE-US-00009 TABLE 8 Neutralization of TNF.alpha.-Induced L929
Cytotoxicity by D2E7 IgG1 Experiment IC.sub.50 [M] 1 1.26 .times.
10.sup.-10 2 1.33 .times. 10.sup.-10 3 1.15 .times. 10.sup.-10
Average 1.25 .+-. 0.01 .times. 10.sup.-10
[0160] This series of experiments confirmed that D2E7, in the
full-length IgG1 form, neutralizes TNF.alpha.-induced L929
cytotoxicity with an average IC.sub.50 [M] of
1.25.+-.0.01.times.10.sup.-10.
B. Inhibition of TNF.alpha. Binding to TNF.alpha. Receptors on
U-937 Cells
[0161] The ability of human anti-hTNF.alpha. antibodies to inhibit
the binding of hTNF.alpha. to hTNF.alpha. receptors on the surface
of cells was examined using the U-937 cell line (ATCC No. CRL
1593), a human histiocytic cell line that expresses hTNF.alpha.
receptors. U-937 cells were grown in RPMI 1640 medium supplemented
with 10% fetal bovine serum (Hyclone A-1111, Hyclone Laboratories,
Logan, Utah), L-glutamine (4 nM), HEPES buffer solution (10 mM),
penicillin (100 .mu.g/ml) and streptomycin (100 .mu.g/ml). To
examine the activity of full-length IgG antibodies, U-937 cells
were preincubated with PBS supplemented with 1 mg/ml of human IgG
(Sigma 1-4506, Sigma Chemical Co., St. Louis, Mo.) for 45 minutes
on ice and then cells were washed three times with binding buffer.
For the receptor binding assay, U-937 cells (5.times.10.sup.6
cells/well) were incubated in a binding buffer (PBS supplemented
with 0.2% bovine serum albumin) in 96-well microtiter plates
(Costar 3799, Costar Corp., Cambridge, Mass.) together with
.sup.125I-labeled rhTNF.alpha. (3.times.10.sup.-10 M; 25 .mu.Ci/ml;
obtained from NEN Research Products, Wilmington, Del.), with or
without anti-hTNF.alpha. antibodies, in a total volume of 0.2 ml.
The plates were incubated on ice for 1.5 hours. Then, 75 .mu.l of
each sample was transferred to 1.0 ml test tubes (Sarstedt 72.700,
Sarstedt Corp., Princeton, N.J.) containing dibutylphthalate (Sigma
D-2270, Sigma Chemical Co., St. Louis, Mo.) and dinonylphthalate
(ICN 210733, ICN, Irvine, Calif.). The test tubes contained a 300
.mu.l mixture of dibutylphthalate and dinonylphthalate, 2:1 volume
ratio, respectively. Free (i.e., unbound) .sup.125I-labeled
rhTNF.alpha. was removed by microcentrifugation for five minutes.
Then, each test tube end containing a cell pellet was cut with the
aid of a microtube scissor (Bel-Art 210180001, Bel-Art Products,
Pequannock, N.J.). The cell pellet contains .sup.125I-labeled
rhTNF.alpha. bound to the p60 or p80 TNF.alpha. receptor, whereas
the aqueous phase above the oil mixture contains excess free
.sup.125I-labeled rhTNF.alpha.. All cell pellets were collected in
a counting tube (Falcon 2052, Becton Dickinson Labware, Lincoln
Park, N.J.) and counted in a scintillation counter.
[0162] Representative results are shown in FIG. 4. The IC.sub.50
value for D2E7 inhibition of hTNF.alpha. binding to hTNF.alpha.
receptors on U-937 cells is approximately 3.times.10.sup.-10 M in
these experiments. These results demonstrate that the D2E7 human
anti-hTNF.alpha. antibody inhibits rhTNF.alpha. binding to
hTNF.alpha. receptors on U-937 cells at concentrations
approximately equivalent to that of the murine anti-hTNF.alpha. mAb
MAK 195.
[0163] In another series of experiments, the ability of the IgG1
form of D2E7 to inhibit rhTNF.alpha. binding to hTNF.alpha.
receptors on U-937 cells was examined as described above. The
results from three independent experiments, and the average
thereof, are summarized below in Table 9:
TABLE-US-00010 TABLE 9 Inhibition of TNF Receptor Binding on U-937
Cells by D2E7 IgG1 Experiment IC.sub.50 [M] 1 1.70 .times.
10.sup.-10 2 1.49 .times. 10.sup.-10 3 1.50 .times. 10.sup.-10
Average 1.56 .+-. 0.12 .times. 10.sup.-10
[0164] This series of experiments confirmed that D2E7, in the
full-length IgG1 form, inhibits TNF receptor binding on U-937 cells
with an average IC.sub.50 [M] of 1.56.+-.0.12.times.10.sup.-10.
[0165] To investigate the inhibitory potency of D2E7 in the binding
of .sup.125I-rhTNF binding to individual p55 and p75 receptors, a
solid phase radioimmunoassay was performed. To measure the
IC.sub.50 values of D2E7 for separate TNF receptors, varying
concentrations of the antibody were incubated with
3.times.10.sup.-10 concentration of .sup.125I-rhTNF. The mixture
was then tested on separate plates containing either the p55 or the
p75 TNF receptors in a dose dependent manner. The results are
summarized below in Table 10:
TABLE-US-00011 TABLE 10 Inhibition of TNF Receptor Binding to p55
and p75 TNFR by D2E7 IgG1 IC.sub.50 [M] Reagent p55 TNFR p 75TNFR
D2E7 1.47 .times. 10.sup.-9 1.26 .times. 10.sup.-9 rhTNF 2.31
.times. 10.sup.-9 2.70 .times. 10.sup.-9
Inhibition of .sup.125I-rhTNF binding to the p55 and p75 TNF
receptors on U937 cells by D2E7 followed a simple sigmoidal curve,
indicating similar IC.sub.50 values for each receptor. In the solid
phase radioimmunoassay (RIA) experiments with recombinant TNF
receptors, IC.sub.50 values for inhibition of .sup.125I-rhTNF
binding to the p55 and the p75 receptors by D2E7 were calculated as
1.47.times.10.sup.-9 and 1.26.times.10.sup.-9 M, respectively. The
decrease in IC.sub.50 values in the solid phase was probably due to
higher density of receptors in the RIA format, as unlabeled rhTNF
also inhibited with similar IC.sub.50 values. The IC.sub.50 values
for inhibition of .sup.125I-rhTNF binding to the p55 and the p75
receptors by unlabeled rhTNF were 2.31.times.10.sup.-9 and
2.70.times.10.sup.-9 M, respectively
C. Inhibition of ELAM-1 Expression on HUVEC
[0166] Human umbilical vein endothelial cells (HUVEC) can be
induced to express endothelial cell leukocyte adhesion molecule 1
(ELAM-1) on their cell-surface by treatment with rhTNF.alpha.,
which can be detected by reacting rhTNF.alpha.-treated HUVEC with
an mouse anti-human ELAM-1 antibody. The ability of human
anti-hTNF.alpha. antibodies to inhibit this TNF.alpha.-induced
expression of ELAM-1 on HUVEC was examined as follows: HUVEC (ATCC
No. CRL 1730) were plated in 96-well plates (5.times.10.sup.4
cells/well) and incubated overnight at 37.degree. C. The following
day, serial dilutions of human anti-hTNF.alpha. antibody (1:10)
were prepared in a microtiter plate, starting with 20-100 .mu.g/ml
of antibody. A stock solution of rhTNF.alpha. was prepared at 4.5
ng/ml, aliquots of rhTNF.alpha. were added to each
antibody-containing well and the contents were mixed well. Controls
included medium alone, medium plus anti-hTNF.alpha. antibody and
medium plus rhTNF.alpha.. The HUVEC plates were removed from their
overnight incubation at 37.degree. C. and the medium gently
aspirated from each well. Two hundred microliters of the
antibody-rhTNF.alpha. mixture were transferred to each well of the
HUVEC plates. The HUVEC plates were then further incubated at
37.degree. C. for 4 hours. Next, a murine anti-ELAM-1 antibody
stock was diluted 1:1000 in RPMI. The medium in each well of the
HUVEC plate was gently aspirated, 50 .mu.l/well of the anti-ELAM-1
antibody solution was added and the HUVEC plates were incubated 60
minutes at room temperature. An .sup.125I-labeled anti-mouse Ig
antibody solution was prepared in RPMI (approximately 50,000 cpm in
500. The medium in each well of the HUVEC plates was gently
aspirated, the wells were washed twice with RPMI and 50 .mu.l of
the .sup.125I-labeled anti-mouse Ig solution was added to each
well. The plates were incubated for one hour at room temperature
and then each well was washed three times with RPMI. One hundred
eighty microliters of 5% SDS was added to each well to lyse the
cells. The cell lysate from each well was then transferred to a
tube and counted in a scintillation counter.
[0167] Representative results are shown in FIG. 5. The IC.sub.50
value for D2E7 inhibition of hTNF.alpha.-induced expression of
ELAM-1 on HUVEC is approximately 6.times.10.sup.-11 M in these
experiments. These results demonstrate that the D2E7 human
anti-hTNF.alpha. antibody inhibits the hTNF.alpha.-induced
expression of ELAM-1 on HUVEC at concentrations approximately
equivalent to that of the murine anti-hTNF.alpha. mAb MAK 195.
[0168] In another series of experiments, the ability of the IgG1
form of D2E7 to inhibit hTNF.alpha.-induced expression of ELAM-1 on
HUVEC was examined as described above. The results from three
independent experiments, and the average thereof, are summarized
below in Table 11:
TABLE-US-00012 TABLE 11 Inhibition of TNF.alpha.-Induced ELAM-1
Expression by D2E7 IgG1 Receptor Experiment IC.sub.50 [M] 1 1.95
.times. 10.sup.-10 2 1.69 .times. 10.sup.-10 3 1.90 .times.
10.sup.-10 Average 1.85 .+-. 0.14 .times. 10.sup.-10
[0169] This series of experiments confirmed that D2E7, in the
full-length IgG1 form, inhibits TNF.alpha.-induced ELAM-1
expression on HUVEC with an average IC.sub.50 NI of
1.85.+-.0.14.times.10.sup.-10.
[0170] The neutralization potency of D2E7 IgG1 was also examined
for the rhTNF induced expression of two other adhesion molecules,
ICAM-1 and VCAM-1. Since the rhTNF titration curve for ICAM-1
expression at 16 hours was very similar to the curve of ELAM-1
expression, the same concentration of rhTNF was used in the
antibody neutralization experiments. The HUVEC were incubated with
rhTNF in the presence of varying concentrations of D2E7 in a
37.degree. C. CO.sub.2 incubator for 16 hours, and the ICAM-1
expression was measured by mouse anti-ICAM-1 antibody followed by
.sup.125I-labeled sheep anti-mouse antibody. Two independent
experiments were performed and the IC.sub.50 values were
calculated. An unrelated human IgG1 antibody did not inhibit the
ICAM-1 expression.
[0171] The experimental procedure to test inhibition of VCAM-1
expression was the same as the procedure for ELAM-1 expression,
except anti-VCAM-1 MAb was used instead of anti-ELAM-1 MAb. Three
independent experiments were performed and the IC.sub.50 values
were calculated. An unrelated human IgG1 antibody did not inhibit
VCAM-1 expression.
[0172] The results are summarized below in Table 12:
TABLE-US-00013 TABLE 12 Inhibition of ICAM-1 and VCAM-1 Expression
by D2E7 IgG1 ICAM-1 Inhibition IC.sub.50 [M] Experiment IC.sub.50
[M] Experiment IC.sub.50 [M] 1 1.84 .times. 10.sup.-10 1 1.03
.times. 10.sup.-10 2 2.49 .times. 10.sup.-10 2 9.26 .times.
10.sup.-11 3 1.06 .times. 10.sup.-10 Average 2.17 .+-. 0.46 .times.
10.sup.-10 Average 1.01 .+-. 0.01 .times. 10.sup.-10
[0173] These experiments demonstrate that treatment of primary
human umbilical vein endothelial cells with rhTNF led to optimum
expression of adhesion molecules: ELAM-1 and VCAM-1 at four hours,
and the maximum up-regulated expression of ICAM-1 at 16 hours. D2E7
was able to inhibit the expression of the three adhesion molecules
in a dose dependent manner. The IC.sub.50 values for the inhibition
of ELAM-1, ICAM-1 and VCAM-1 were 1.85.times.10.sup.-10,
2.17.times.10.sup.-10 and 1.01.times.10.sup.-10 M, respectively.
These values are very similar, indicating similar requirements for
the dose of rhTNF activation signal to induce ELAM-1, ICAM-1 and
VCAM-1 expression. Interestingly, D2E7 was similarly effective in
the longer inhibition assay of the ICAM-1 expression. The ICAM-1
inhibition assay required 16 hours of co-incubation of rhTNF and
D2E7 with HUVEC as opposed to 4 hours required for the ELAM-1 and
the VCAM-1 inhibition assays. Since D2E7 has a slow off-rate for
rhTNF, it is conceivable that during the 16 hour co-incubation
period there was no significant competition by the TNF receptors on
the HUVEC.
D. In Vivo Neutralization of hTNF.alpha.
[0174] Three different in vivo systems were used to demonstrate
that D2E7 is effective at inhibiting hTNF.alpha. activity in
vivo.
[0175] I. Inhibition of TNF-Induced Lethality in
D-Galactosamine-Sensitized Mice
[0176] Injection of recombinant human TNF.alpha. (rhTNF.alpha.) to
D-galactosamine sensitized mice causes lethality within a 24 hour
time period. TNF.alpha. neutralizing agents have been shown to
prevent lethality in this model. To examine the ability of human
anti-hTNF.alpha. antibodies to neutralize hTNF.alpha. in vivo in
this model, C57B1/6 mice were injected with varying concentrations
of D2E7-IgG1, or a control protein, in PBS intraperitoneally
(i.p.). Mice were challenged 30 minutes later with 1 .mu.g of
rhTNF.alpha. and 20 mg of D-galactosamine in PBS i.p., and observed
24 hours later. These amount of rhTNF.alpha. and D-galactosamine
were previously determined to achieve 80-90% lethality in these
mice.
[0177] Representative results, depicted as a bar graph of %
survival versus antibody concentration, are shown in FIG. 6. The
black bars represent D2E7, whereas the hatched bars represent MAK
195. Injection of 2.5-25 .mu.g of D2E7 antibody per mouse protected
the animals from TNF.alpha.-induced lethality. The ED.sub.50 value
is approximately 1-2.5 .mu.g/mouse. The positive control antibody,
MAK 195, was similar in its protective ability. Injection of D2E7
in the absence of rhTNF.alpha. did not have any detrimental effect
on the mice. Injection of a non-specific human IgG1 antibody did
not offer any protection from TNF.alpha.-induced lethality.
[0178] In a second experiment, forty-nine mice were divided into 7
equal groups. Each group received varying doses of D2E7 thirty
minutes prior to receiving an LD.sub.80 dose of
rhTNF/D-galactosamine mixture (1.0 .mu.g rhTNF and 20 mg
D-galactosamine per mouse). Control group 7 received normal human
IgG1 kappa antibody at 25 .mu.g/mouse dose. The mice were examined
24 hours later. Survival for each group is summarized below in
Table 13.
TABLE-US-00014 TABLE 13 24 Hour Survival After Treatment with D2E7
Survival Survival Group (alive/total) (%) 1 (no antibody) 0/7 0 2
(1 .mu.g) 1/7 14 3 (2.6 .mu.g) 5/7 71 4 (5.2 .mu.g) 6/7 86 5 (26
.mu.g) 6/7 86 6 (26 .mu.g; no rhTNF) 7/7 100 7 (25 .mu.g Hu IgG1)
1/7 14
[0179] II. Inhibition of TNF-Induced Rabbit Pyrexia
[0180] The efficacy of D2E7 in inhibiting rhTNF-induced pyrexia
response in rabbits was examined Groups of three NZW female rabbits
weighing approximately 2.5 kg each were injected intravenously with
D2E7, rhTNF, and immune complexes of D2E7 and rhTNF. Rectal
temperatures were measured by thermistor probes on a Kaye thermal
recorder every minute for approximately 4 hours. Recombinant human
TNF in saline, injected at 5 .mu.g/kg, elicited a rise in
temperature greater than 0.4.degree. C. at approximately 45 minutes
after injection. The antibody preparation by itself, in saline at a
dose of 138 .mu.g/kg, did not elicit a rise in temperature in the
rabbits up to 140 minutes after administration. In all further
experiments, D2E7 or control reagents (human IgG1 or a saline
vehicle) were injected i.v. into rabbits followed 15 minutes later
by an injection of rhTNF in saline at 5 .mu.g/kg i.v.
Representative results of several experiments are summarized below
in Table 14:
TABLE-US-00015 TABLE 14 Inhibition of rhTNF-induced Pyrexia with
D2E7 in Rabbits D2E7 Temp. rise*, .degree. C. Peak Temp. dose rhTNF
+ % Molar Ratio minutes (.mu.g/kg) rhTNF D2E7 Inhib.** D2E7:rhTNF
post rhTNF 14 0.53 0.25 53 1 60 24 0.43 0.13 70 1.6 40 48 0.53 0.03
94 3.3 50 137 0.53 0.00 100 9.5 60 792 0.80 0.00 100 55 60 *= Peak
temperature **= % inhibition = (1 - {temperature rise with rhTNF
& D2E7/temperature rise with rhTNF alone}) .times. 100.
Intravenous pretreatment with D2E7 at a dose of 14 .mu.g/kg
partially inhibited the pyrogenic response, compared to rabbits
pre-treated with saline alone. D2E7 administered at 137 .mu.g/kg
totally suppressed the pyrogenic response of rhTNF in the same
experiment. In a second experiment, D2E7 administered at 24
.mu.g/kg also partially suppressed the pyrogenic response, compared
to rabbits pretreated with saline alone. The molar ratio of D2E7 to
rhTNF was 1/6:1 in this experiment. In a third experiment, D2E7
injected i.v. at 48 .mu.g/kg (molar ratio D2E7:rhTNF=3.3:1) totally
suppressed the pyrogenic response, compared to rabbits pretreated
with the control human IgG1 in saline at 30 .mu.g/kg. In the final
experiment, rabbits pretreated with D2E7 (792 .mu.g/kg) at a very
high molar ratio to rhTNF (55:1) did not develop any rise in
temperature at any time up to 4 hours of observation. Treatment of
rabbits with immune complexes generated from a mixture of D2E7 and
rhTNF incubated at 37.degree. C. for 1 hour at a molar ratio of
55:1, without subsequent rhTNF administration, also did not elicit
any rise in temperature in the same experiment.
[0181] III. Prevention of Polyarthritis in Tg197 Transgenic
Mice
[0182] The effect of D2E7 on disease development was investigated
in a transgenic murine model of arthritis. Transgenic mice (Tg197)
have been generated that express human wild type TNF (modified in
the 3' region beyond the coding sequences) and these mice develop
chronic polyarthritis with 100% incidence at 4-7 weeks of age (see
EMBO J. (1991) 10:4025-4031 for further description of the Tg197
model of polyarthritis).
[0183] Transgenic animals were identified by PCR at 3 days of age
Litters of transgenic mice were divided into six groups. Transgenic
mice were verified by slot-blot hybridization analysis at 15 days
of age. The treatment protocols for the six groups were as follows:
Group 1=no treatment; Group 2=saline (vehicle); Group 3=D2E7 at 1.5
jag/g; Group 4=D2E7 at 15 .mu.g/g; Group 5=D2E7 at 30 .mu.g/g; and
Group 6=IgG1 isotype control at 30 .mu.g/g. A litter with non
transgenic mice was also included in the study to serve as a
control (Group 7--nontransgenic; no treatment). Each group received
three i.p. injections per week of the indicated treatments.
Injections continued for 10 weeks. Each week, macroscopic changes
in joint morphology were recorded for each animal. At 10 weeks, all
mice were sacrificed and mouse tissue was collected in formalin.
Microscopic examination of the tissue was performed.
[0184] Animal weight in grams was taken for each mouse at the start
of each week. At the same time measurements of joint size (in mm)
were also taken, as a measurement of disease severity. Joint size
was established as an average of three measurements on the hind
right ankle using a micrometer device. Arthritic scores were
recorded weekly as follows: 0=No arthritis, (normal appearance and
flexion); +=mild arthritis (joint distortion); ++=moderate
arthritis (swelling, joint deformation) and +++=heavy arthritis
(ankylosis detected on flexion and severely impaired movement).
Histopathological scoring based on haematoxylin/eosin staining of
joint sections was based as follows; 0=No detectable disease;
1=proliferation of the synovial membrane; 2=heavy synovial
thickening 3=cartilage destruction and bone erosion.
[0185] The effect of D2E7 treatment on the mean joint size of the
Tg197 transgenic arthritic mice is shown in the graph of FIG. 9.
The histopathological and arthritic scores of the Tg197 transgenic
mice, at 11 weeks of age, are summarized below in Table 15:
TABLE-US-00016 TABLE 15 Effect of D2E7 on Histopathology and
Arthritic Score in Tg197 Mice Group Treatment Histopathological
Score Arthritic Score 1 none .sup. 3 (7/70 +++ (7/7) 2 saline 3
(8/8) +++ (8/8) 6 IgG1 control 3 (9/9) +++ (7/9) 3 D2E7 at 1.5
.mu.g/g 0 (6/8) 0 (8/8) 4 D2E7 at 15 .mu.g/g 0 (7/8) 0 (8/8) 5 D2E7
at 30 .mu.g/g 0 (8/8) 0 (8/8)
[0186] This experiment demonstrated that the D2E7 antibody has a
definite beneficial effect on transgenic mice expressing the
wild-type human TNF (Tg197) with no arthritis evident after the
study period.
E. D2E7 Neutralization of TNF.alpha.s from Other Species
[0187] The binding specificity of D2E7 was examined by measuring
its ability to neutralize tumor necrosis factors from various
primate species and from mouse, using an L929 cytotoxicity assay
(as described in Example 4, subsection A, above). The results are
summarized in Table 16 below:
TABLE-US-00017 TABLE 16 Ability of D2E7 to Neutralize TNF from
Different Species in the L929 Assay IC.sub.50 for D2E7 TNF.alpha.*
Source Neutralization (M)** Human Recombinant 7.8 .times.
10.sup.-11 Chimpanzee LPS-stimulated PBMC 5.5 .times. 10.sup.-11
baboon Recombinant 6.0 .times. 10.sup.-11 marmoset LPS-stimulated
PBMC 4.0 .times. 10.sup.-10 cynomolgus LPS-stimulated PBMC 8.0
.times. 10.sup.-11 rhesus LPS-stimulated PBMC 3.0 .times.
10.sup.-11 canine LPS-stimulated WBC 2.2 .times. 10.sup.-10 porcine
Recombinant 1.0 .times. 10.sup.-7 murine Recombinant >1.0
.times. 10.sup.-7
[0188] The results in Table 16 demonstrate that D2E7 can neutralize
the activity of five primate TNF.alpha.s approximately equivalently
to human TNF.alpha. and, moreover, can neutralize the activity of
canine TNF.alpha. (about ten-fold less well than human TNF.alpha.)
and porcine and mouse TNF.alpha. (about .about.1000-fold less well
than human TNF.alpha.). Moreover, the binding of D2E7 to solution
phase rhTNF.alpha. was not inhibited by other cytokines, such as
lymphotoxin (TNF.beta.), IL-1.alpha., IL-1.beta., IL-2, IL-4, IL-6,
IL-8, IFN.gamma. and TGF.beta., indicating that D2E7 is very
specific for its ligand TNF.alpha..
F. Lack of Cytokine Release by Human Whole Blood Incubated with
D2E7
[0189] In this example, the ability of D2E7 to induce, by itself,
normal human blood cells to secrete cytokines or shed cell surface
molecules was examined. D2E7 was incubated with diluted whole blood
from three different normal donors at varying concentrations for 24
hours. An LPS positive control was run at the same time, at a
concentration previously determined to stimulate immunocompetent
blood cells to secrete cytokines. The supernatants were harvested
and tested in a panel of ten soluble cytokine, receptor and
adhesion molecule ELISA kits: IL-1a, IL-113, IL-1 receptor
antagonist, IL-6, IL-8, TNF.alpha., soluble TNF receptor I, soluble
TNF receptor II, soluble ICAM-1 and soluble E-selectin. No
significant amounts of cytokines or shed cell surface molecules
were measured as a result of D2E7 antibody co-incubation, at
concentrations up to 343 .mu.g/ml. Control cultures without the
addition of the antibody also did not yield any measurable amounts
of cytokines, whereas the LPS co-culture control yielded elevated
values in the high picogram to low nanogram range. These results
indicate that D2E7 did not induce whole blood cells to secrete
cytokines or shed cell surface proteins above normal levels in ex
vivo cultures.
[0190] Forming part of the present disclosure is the appended
Sequence Listing, the contents of which are summarized in the table
below:
TABLE-US-00018 ANTIBODY SEQ ID NO: CHAIN REGION SEQUENCE TYPE 1
D2E7 VL amino acid 2 D2E7 VH amino acid 3 D2E7 VL CDR3 amino acid 4
D2E7 VH CDR3 amino acid 5 D2E7 VL CDR2 amino acid 6 D2E7 VH CDR2
amino acid 7 D2E7 VL CDR1 amino acid 8 D2E7 VH CDR1 amino acid 9
2SD4 VL amino acid 10 2SD4 VH amino acid 11 2SD4 VL CDR3 amino acid
12 EP B12 VL CDR3 amino acid 13 VL10E4 VL CDR3 amino acid 14
VL100A9 VL CDR3 amino acid 15 VLL100D2 VL CDR3 amino acid 16 VLL0F4
VL CDR3 amino acid 17 LOE5 VL CDR3 amino acid 18 VLLOG7 VL CDR3
amino acid 19 VLLOG9 VL CDR3 amino acid 20 VLLOH1 VL CDR3 amino
acid 21 VLLOH10 VL CDR3 amino acid 22 VL1B7 VL CDR3 amino acid 23
VL1C1 VL CDR3 amino acid 24 VL0.1F4 VL CDR3 amino acid 25 VL0.1H8
VL CDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 VH CDR3
amino acid 28 VH1B11 VH CDR3 amino acid 29 VH1D8 VH CDR3 amino acid
30 VH1A11 VH CDR3 amino acid 31 VH1B12 VH CDR3 amino acid 32 VH1E4
VH CDR3 amino acid 33 VH1F6 VH CDR3 amino acid 34 3C-H2 VH CDR3
amino acid 35 VH1-D2.N VH CDR3 amino acid 36 D2E7 VL nucleic acid
37 D2E7 VH nucleic acid
EQUIVALENTS
[0191] 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
371107PRTArtificialD2E7 light chain variable region 1Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg
Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 2121PRTArtificialD2E7 heavy chain variable
region 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His
Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val
Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 39PRTArtificialD2E7 light
chain variable region CDR3 3Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa1 5
412PRTArtificialD2E7 heavy chain variable region CDR3 4Val Ser Tyr
Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5 10 57PRTArtificialD2E7 light
chain variable region CDR2 5Ala Ala Ser Thr Leu Gln Ser1 5
617PRTArtificialD2E7 heavy chain variable region CDR2 6Ala Ile Thr
Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu1 5 10 15
Gly711PRTArtificialD2E7 light chain variable region CDR1 7Arg Ala
Ser Gln Gly Ile Arg Asn Tyr Leu Ala1 5 10 85PRTArtificialD2E7 heavy
chain variable region CDR1 8Asp Tyr Ala Met His1 5
9107PRTArtificial2SD4 light chain variable region 9Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr
Asn Ser Ala Pro Tyr 85 90 95 Ala Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 10121PRTArtificial2SD4 heavy chain variable region
10Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Asp Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp
Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Ala Val Ser Arg Asp
Asn Ala Lys Asn Ala Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Ala Ser Tyr
Leu Ser Thr Ser Ser Ser Leu Asp Asn Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 119PRTArtificial2SD4 light chain
variable region CDR3 11Gln Lys Tyr Asn Ser Ala Pro Tyr Ala1 5
129PRTArtificialEP B12 light chain variable region CDR3 12Gln Lys
Tyr Asn Arg Ala Pro Tyr Ala1 5 139PRTArtificialVL10E4 light chain
variable region CDR3 13Gln Lys Tyr Gln Arg Ala Pro Tyr Thr1 5
149PRTArtificialVL100A9 light chain variable region CDR3 14Gln Lys
Tyr Ser Ser Ala Pro Tyr Thr1 5 159PRTArtificialVLL100D2 light chain
variable region CDR3 15Gln Lys Tyr Asn Ser Ala Pro Tyr Thr1 5
169PRTArtificialVLL0F4 light chain variable region CDR3 16Gln Lys
Tyr Asn Arg Ala Pro Tyr Thr1 5 179PRTArtificialLOE5 light chain
variable region CDR3 17Gln Lys Tyr Asn Ser Ala Pro Tyr Tyr1 5
189PRTArtificialVLLOG7 light chain variable region CDR3 18Gln Lys
Tyr Asn Ser Ala Pro Tyr Asn1 5 199PRTArtificialVLLOG9 light chain
variable region CDR3 19Gln Lys Tyr Thr Ser Ala Pro Tyr Thr1 5
209PRTArtificialVLLOH1 light chain variable region CDR3 20Gln Lys
Tyr Asn Arg Ala Pro Tyr Asn1 5 219PRTArtificialVLLOH10 light chain
variable region CDR3 21Gln Lys Tyr Asn Ser Ala Ala Tyr Ser1 5
229PRTArtificialVL1B7 light chain variable region CDR3 22Gln Gln
Tyr Asn Ser Ala Pro Asp Thr1 5 239PRTArtificialVL1C1 light chain
variable region CDR3 23Gln Lys Tyr Asn Ser Asp Pro Tyr Thr1 5
249PRTArtificialVL0.1F4 light chain variable region CDR3 24Gln Lys
Tyr Ile Ser Ala Pro Tyr Thr1 5 259PRTArtificialVL0.1H8 light chain
variable region CDR3 25Gln Lys Tyr Asn Arg Pro Pro Tyr Thr1 5
269PRTArtificialLOE7.A light chain variable region CDR3 26Gln Arg
Tyr Asn Arg Ala Pro Tyr Ala1 5 2712PRTArtificial2SD4 heavy chain
variable region CDR3 27Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp
Asn1 5 10 2812PRTArtificialVH1B11 heavy chain variable region CDR3
28Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys1 5 10
2912PRTArtificialVH1D8 heavy chain variable region CDR3 29Ala Ser
Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr1 5 10
3012PRTArtificialVH1A11 heavy chain variable region CDR3 30Ala Ser
Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp1 5 10
3112PRTArtificialVH1B12 heavy chain variable region CDR3 31Ala Ser
Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr1 5 10
3212PRTArtificialVH1E4 heavy chain variable region CDR3 32Ala Ser
Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr1 5 10
3312PRTArtificialVH1F6 heavy chain variable region CDR3 33Ala Ser
Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr1 5 10
3412PRTArtificial3C-H2 heavy chain variable region CDR3 34Ala Ser
Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr1 5 10
3512PRTArtificialVH1-D2.N heavy chain variable region CDR3 35Val
Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn1 5 10
36321DNAArtificialD2E7 light chain variable region 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
32137363DNAArtificialD2E7 heavy chain variable region 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
* * * * *