U.S. patent application number 13/775540 was filed with the patent office on 2013-11-07 for il-1 binding proteins.
The applicant listed for this patent is Dominic J. Ambrosi, Chung-ming Hsieh, Renee Miller, Chengbin Wu. Invention is credited to Dominic J. Ambrosi, Chung-ming Hsieh, Renee Miller, Chengbin Wu.
Application Number | 20130295004 13/775540 |
Document ID | / |
Family ID | 43876580 |
Filed Date | 2013-11-07 |
United States Patent
Application |
20130295004 |
Kind Code |
A1 |
Hsieh; Chung-ming ; et
al. |
November 7, 2013 |
IL-1 BINDING PROTEINS
Abstract
The present invention describes IL-1.beta. binding proteins,
including chimeric, CDR-grafted, and humanized antibodies that bind
IL-1.beta.. Binding proteins of the invention have high affinity
for IL-1.beta. and neutralize IL-1.beta. activity. A binding
protein of the invention can be a full-length antibody or an
IL-1.beta.-binding portion thereof. Methods of making and methods
of using the binding proteins of the invention are also described.
The IL-1.beta. binding proteins of the invention are useful for
detecting IL-1.beta. and for inhibiting IL-1.beta. activity,
including in a human subject suffering from a disease or disorder
in which IL-1.beta. activity is detrimental.
Inventors: |
Hsieh; Chung-ming; (Newton,
MA) ; Wu; Chengbin; (Shanghai, CN) ; Miller;
Renee; (North Grosvenordale, CT) ; Ambrosi; Dominic
J.; (Shrewsbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hsieh; Chung-ming
Wu; Chengbin
Miller; Renee
Ambrosi; Dominic J. |
Newton
Shanghai
North Grosvenordale
Shrewsbury |
MA
CT
MA |
US
CN
US
US |
|
|
Family ID: |
43876580 |
Appl. No.: |
13/775540 |
Filed: |
February 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12905499 |
Oct 15, 2010 |
8398966 |
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13775540 |
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61251856 |
Oct 15, 2009 |
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Current U.S.
Class: |
424/1.11 ;
424/139.1; 435/188; 530/387.3; 530/387.9; 530/391.3; 530/391.7 |
Current CPC
Class: |
A61P 25/04 20180101;
A61P 1/04 20180101; A61P 33/00 20180101; A61P 31/20 20180101; A61P
9/06 20180101; A61P 17/14 20180101; A61P 31/16 20180101; A61P 31/18
20180101; A61P 11/00 20180101; A61P 11/06 20180101; C07K 2317/92
20130101; A61P 35/02 20180101; C07K 2317/56 20130101; A61P 21/00
20180101; A61P 25/18 20180101; A61K 39/3955 20130101; A61P 9/12
20180101; A61P 15/00 20180101; A61P 35/00 20180101; A61P 37/02
20180101; C12N 9/96 20130101; A61P 13/12 20180101; A61P 25/16
20180101; A61P 3/10 20180101; A61P 7/00 20180101; A61P 7/06
20180101; A61P 43/00 20180101; C07K 2317/24 20130101; A61P 9/10
20180101; A61P 5/14 20180101; A61P 29/00 20180101; A61P 9/00
20180101; A61P 17/06 20180101; C07K 16/245 20130101; A61P 17/04
20180101; A61P 5/18 20180101; A61P 15/08 20180101; A61P 25/28
20180101; A61P 17/00 20180101; A61P 17/02 20180101; A61P 19/02
20180101; A61P 25/00 20180101; A61P 31/00 20180101; A61P 25/14
20180101; A61P 31/14 20180101; A61P 1/16 20180101; A61P 27/02
20180101; A61P 31/04 20180101; A61P 9/08 20180101; G01N 2333/545
20130101; A61P 1/00 20180101; A61P 1/18 20180101; A61P 21/04
20180101; A61P 33/06 20180101; A61P 25/24 20180101; A61P 37/08
20180101; C07K 2317/76 20130101; A61P 3/00 20180101; A61P 7/02
20180101; A61P 11/02 20180101; A61P 9/04 20180101; A61P 25/32
20180101; C07K 2317/565 20130101; A61K 45/06 20130101; A61P 31/10
20180101; A61P 37/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
424/1.11 ;
530/387.9; 530/387.3; 530/391.3; 530/391.7; 435/188; 424/139.1 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; C12N 9/96 20060101 C12N009/96 |
Claims
1. (canceled)
2. A binding protein that is capable of binding to IL-1.beta.,
wherein said binding protein comprises at least one complementarity
determining region (CDR) comprising an amino acid sequence selected
from the group consisting of residues 31-35 of SEQ ID NO:26
(CDR-H1); residues 50-65 of SEQ ID NO:26 (CDR-H2); residues 98-111
of SEQ ID NO:26 (CDR-H3); residues 24-34 of SEQ ID NO:27 (CDR-L1);
residues 50-56 of SEQ ID NO:27 (CDR-L2); and residues 89-97 of SEQ
ID NO:27 (CDR-L3).
3-6. (canceled)
7. The binding protein of claim 2, further comprising a human
acceptor framework sequence.
8. The binding protein of claim 7, wherein the human acceptor
framework sequence comprises an amino acid sequence selected from
the group consisting of SEQ ID NOS:10-25.
9. The binding protein of claim 8, wherein said human acceptor
framework sequence comprises at least one amino acid substitution,
wherein the amino acid sequence of the human acceptor framework
sequence is at least 60% identical to an amino acid sequence of any
one of SEQ ID NOs:10-25.
10-13. (canceled)
14. The binding protein of claim 2, wherein said binding protein
has greater than 90% identity to a binding protein comprising a
variable heavy chain polypeptide and a variable light chain
polypeptide comprising the amino acid sequences of SEQ ID NO: 30
and SEQ ID NO: 34, respectively.
15. The binding protein of claim 2, wherein said binding protein is
selected from the group consisting of: an immunoglobulin molecule,
a disulfide linked Fv, a monoclonal antibody, an scFv, a chimeric
antibody, a single domain antibody, a CDR-grafted antibody, a
diabody, a humanized antibody, a multispecific antibody, an Fab, a
dual specific antibody, a DVD-Ig protein, a Fab', a bispecific
antibody, a F(ab')2, and a Fv.
16. The binding protein of claim 2, wherein said binding protein
comprises a heavy chain immunoglobulin constant domain selected
from the group consisting of a human IgM constant domain, a human
IgG4 constant domain, a human IgG1 constant domain, a human IgE
constant domain, a human IgG2 constant domain, a human IgG3
constant domain, and a human IgA constant domain.
17. The binding protein of claim 2, further comprising a heavy
chain constant region having an amino acid sequence selected from
the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
18. The binding protein of claim 2, further comprising a light
chain constant region having an amino acid sequence selected from
the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.
19. (canceled)
20. The binding protein of claim 2, wherein said binding protein is
capable of neutralizing human IL-1.beta..
21-24. (canceled)
25. The binding protein of claim 2, wherein said binding protein
further comprises an agent selected from the group consisting of:
an immunoadhesion molecule, an imaging agent, a therapeutic agent,
and a cytotoxic agent.
26. The binding protein of claim 25, wherein said agent is an
imaging agent selected from the group consisting of: a radiolabel,
an enzyme, a fluorescent label, a luminescent label, a
bioluminescent label, a magnetic label, and biotin.
27. The binding protein of claim 25, wherein said imaging agent is
a radiolabel selected from the group consisting of: 3H, 14C, 35S,
90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm.
28. The binding protein of claim 25, wherein said agent is a
therapeutic or cytotoxic agent selected from the group consisting
of: an anti-metabolite, an alkylating agent, an antibiotic, a
growth factor, a cytokine, an anti-angiogenic agent, an
anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent.
29-53. (canceled)
54. A pharmaceutical composition comprising the binding protein of
claim 2, and a pharmaceutically acceptable carrier.
55. The pharmaceutical composition of claim 54, wherein said
pharmaceutically acceptable carrier functions as an adjuvant useful
to increase the absorption, or dispersion of said binding
protein.
56. The pharmaceutical composition of claim 55, wherein said
adjuvant is hyaluronidase.
57. The pharmaceutical composition of claim 54, further comprising
at least one additional agent for treating a disorder in which
IL-1.beta. activity is detrimental.
58. The pharmaceutical composition of claim 57, wherein said
additional agent is selected from the group consisting of: a
therapeutic agent; an imaging agent; a cytotoxic agent; an
angiogenesis inhibitor; a kinase inhibitor; a co-stimulation
molecule blocker; an adhesion molecule blocker; an anti-cytokine
antibody or functional fragment thereof; methotrexate; cyclosporin;
rapamycin; FK506; a detectable label or reporter; a TNF antagonist;
an anti-rheumatic; a muscle relaxant; a narcotic; a non-steroid
anti-inflammatory drug (NSAID); an analgesic; an anesthetic; a
sedative; a local anesthetic; a neuromuscular blocker; an
antimicrobial; an antipsoriatic; a corticosteroid; an anabolic
steroid; an erythropoietin; an immunization; an immunoglobulin; an
immunosuppressive; a growth hormone; a hormone replacement drug; a
radiopharmaceutical; an antidepressant; an antipsychotic; a
stimulant; an asthma medication; a beta agonist; an inhaled
steroid; an oral steroid; an epinephrine or analog thereof; a
cytokine; and a cytokine antagonist.
59-67. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 61/251,856 filed Oct. 15, 2009, the
entire contents of which is incorporated herein by reference for
any purpose.
FIELD OF THE INVENTION
[0002] The present invention relates to IL-1 binding proteins, and
specifically to their uses in the prevention and/or treatment of
IL-1 mediated diseases.
BACKGROUND OF THE INVENTION
[0003] Cytokines, such as interleukin-1 (IL-1) and tumor necrosis
factor (TNF), are molecules produced by a variety of cells, such as
monocytes and macrophages, which are mediators of inflammatory
processes. Interleukin-1 is a cytokine with a wide range of
biological and physiological effects, including fever,
prostaglandin synthesis (in, e.g., fibroblasts, muscle cells and
endothelial cells), T-lymphocyte activation, and interleukin-2
production.
[0004] The Interleukin-1 superfamily: The original members of the
IL-1 superfamily are IL-1.alpha., IL-1.beta., and the IL-1 Receptor
antagonist (IL-1RA). IL-1.alpha. and -.beta. are pro-inflammatory
cytokines involved in immune defense against infection. The
IL-1R.alpha. is a molecule that competes for receptor binding with
IL-1.alpha. and IL-1.beta., blocking their role in immune
activation. Recent years have seen the addition of other molecules
to the IL-1 superfamily including IL-18 (see Dinarello (1994) FASEB
J. 8(15):1314-3225; Huising et al. (2004) Dev. Comp. Immunol.
28(5):395-413) and six more genes with structural homology to
IL-1.alpha., IL-1.beta. or IL-1RA. These latter six members are
named IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, and IL1F10. In accordance,
IL-1.alpha., IL-1.beta., and IL-1RA have been renamed IL-1F1,
IL-1F2, and IL-1F3, respectively (see Sims et al. (2001) Trends
Immunol. 22(10):536-537; Dunn et al. (2001) Trends Immunol.
22(10):533-536). A further putative member of the IL-1 family has
been described called IL-33 or IL-1F11, although this name is not
officially accepted in the HGNC gene family nomenclature
database.
[0005] Both IL-1.alpha. and IL-1.beta. are produced by macrophages,
monocytes and dendritic cells. They form an important part of the
inflammatory response of the body against infection. These
cytokines increase the expression of adhesion factors on
endothelial cells to enable transmigration of leukocytes, the cells
that fight pathogens, to sites of infection and re-set the
hypothalamus thermoregulatory center, leading to an increased body
temperature which expresses itself as fever. IL-1 is therefore
calledan endogenous pyrogen. The increased body temperature helps
the body's immune system to fight infection. IL-1 is also important
in the regulation of hematopoiesis. IL-1.alpha. is a pleiotropic
cytokine involved in various immune responses, inflammatory
processes, and hematopoiesis. IL-1.alpha. is produced by activated
macrophages, stimulates thymocyte proliferation by inducing IL-2
release, B-cell maturation and proliferation, and fibroblast growth
factor activity. IL-1.alpha. proteins are involved in the
inflammatory response, being identified as endogenous pyrogens, and
are reported to stimulate the release of prostaglandin and
collagenase from synovial cells. It is produced as a proprotein
that is proteolytically processed by calpain and released in a
mechanism that is still not well studied. This gene and eight other
interleukin 1 family genes form a cytokine gene cluster on
chromosome 2. IL-1.alpha. and its disease-causing effects are
described in detail in Ibelgaufts, Lexikon Zytokine (Cytokine
Dictionary), Medikon Verlag, Munich 1992, and in the literature
cited therein. Reference is also made to the undesirable effects of
IL-1.alpha. in, for example, Oppenheim et al. (1986) Immunol. Today
7:45-56, Durum et al. (1985) Ann. Rev. Immunol. 3:263-287 and
Synnons et al. (1989) Lymphokine Res. 8:365-372. IL-1.alpha. was
originally termed "catabolin" because of its effect in increasing
cartilage resorption, but also as "monocyte cell factor" (MCF)
because of its stimulatory effect on collagenase and prostaglandin
in synovial cells, and as "leucocyte endogenous factor" (LEM)
having a stimulatory effect on acute phase reactions. In addition,
IL-1.alpha. has a broad spectrum of biological activities, since
IL-1.alpha. is synthesized by many different cells, such as
monocytes, macrophages, fibroblasts, endothelial cells and
lymphocytes, and many cells possess specific receptors for
IL-1.alpha.. IL-1.alpha. therefore occupies a central position as
the trigger for various disorders and symptoms of disorders. These
disorders are often predominantly serious disorders for which there
is little or no treatment. It has been suggested that the
polymorphism of these genes is associated with rheumatoid arthritis
and Alzheimer's disease. IL-1 in general has been implicated in
many human diseases, including arthritis, pulmonary fibrosis,
diseases of the central nervous system, diabetes mellitus, and
certain cardiovascular diseases.
[0006] IL-1.beta. production in peripheral tissue has also been
associated with hyperalgesia (increased sensitivity to pain)
associated with fever (Morgan et al. (2004) Brain Res.
1022(1-2):96-100). For the most part, these two forms of IL-1 bind
to the same cellular receptor. This receptor is composed of two
related, but non-identical, subunits that transmit intracellular
signals via a pathway that is mostly shared with certain other
receptors. These include the Toll family of innate immune receptors
and the receptor for IL-18. IL-1.alpha. and IL-1.beta. also possess
similar biological properties, including induction of fever, slow
wave sleep, and neutrophilia, T- and B-lymphocyte activation,
fibroblast proliferation, cytotoxicity for certain cells, induction
of collagenases, synthesis of hepatic acute phase proteins, and
increased production of colony stimulating factors and
collagen.
[0007] cDNAs encoding the two distinct forms of IL-1 have been
isolated and expressed; these cDNAs represent two different gene
products, termed IL-1.beta. (Auron et al. (1984) Proc. Natl. Acad.
Sci. USA 81:7909) and IL-1.alpha. (Lomedico et al. (1984) Nature
312:458). IL-1.beta. is the predominant form produced by human
monocytes both at the mRNA and protein levels. The two forms of
human IL-1 share only 26% amino acid homology. Despite their
distinct polypeptide sequences, the two forms of IL-1 have
structural similarities (Auron et al. (1985) J. Mol. Cell. Immunol.
2:169), in that the amino acid homology is confined to discrete
regions of the IL-1 molecule.
[0008] IL-1.alpha. and IL-1.beta. are produced as precursor
peptides. In other words they are made as a long protein that is
then processed to release a shorter, active molecule, which is
called the mature protein. Mature IL-1.beta., for example, is
released from Pro-IL-1.beta. following cleavage by a certain member
of the caspase family of proteins, called caspase-1 or the
interleukin-1 converting enzyme (ICE). The 3-dimensional structure
of the mature forms of each member of the human IL-1 superfamily is
composed of 12-14 .beta.-strands producing a barrel-shaped
protein.
[0009] There is a need in the art for improved antibodies that bind
IL-1.beta. for use in new therapies against IL-1.beta. associated
diseases and for use in detecting IL-1.beta. in samples and
tissues.
SUMMARY OF THE INVENTION
[0010] The present invention provides a novel family of binding
proteins, including monoclonal antibodies (mAbs), CDR grafted
antibodies, humanized antibodies, affinity matured antibodies, and
fragments thereof, capable binding human IL-1.beta., binding with
high affinity, and binding and neutralizing IL-1.beta.. The
invention provides a therapeutic means with which to inhibit human
IL-1.beta. and provides compositions and methods for treating
diseases and disorders associated with increased levels of
IL-1.beta. particularly inflammatory disorders. The invention also
provides an antibody, or antigen binding portion thereof, capable
of binding IL-1.beta. wherein said antibody, or antigen binding
portion thereof, capable of binding IL-1.beta. comprises an amino
acid sequence selected from the group consisting of 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, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40. In another
embodiment, the invention provides means for detecting and/or
measuring human IL-1.beta. in samples, mixtures, and tissues.
[0011] In one aspect of the invention, the isolated binding protein
is capable of binding human IL-1.beta. and comprises at least one
CDR comprising an amino acid sequence selected from the group
consisting of residues 31-35 of SEQ ID NO:26 (CDR-H1); residues
50-65 of SEQ ID NO:26 (CDR-H2); residues 98-111 of SEQ ID NO:26
(CDR-H3); residues 24-34 of SEQ ID NO:27 (CDR-L1); residues 50-56
of SEQ ID NO:27 (CDR-L2); and residues 89-97 of SEQ ID NO:27
(CDR-L3).
[0012] In another embodiment, a binding protein comprises at least
three CDRs that are selected from a variable domain CDR set,
wherein the variable domain CDR set is selected from the group
consisting of a VH CDR Set comprising residues 31-35 of SEQ ID
NO:26 (CDR-H1); residues 50-65 of SEQ ID NO:26 (CDR-H2); and
residues 98-111 of SEQ ID NO:26 (CDR-H3); and a VL CDR Set
comprising residues 24-34 of SEQ ID NO:27 (CDR-L1); residues 50-56
of SEQ ID NO:27 (CDR-L2); and residues 89-97 of SEQ ID NO:27
(CDR-L3).
[0013] In an embodiment, a binding protein comprises a variable
heavy chain (VH) set of three CDRs and also comprises a variable
light chain (VL) set of three CDRS.
[0014] In another embodiment of the invention, an IL-1.beta.
binding protein comprising one or more CDRs described above further
comprises a corresponding human heavy chain acceptor framework
sequence (for CDR-H1, CDR-H2, and CDR-H3 sequences) and/or a
corresponding human light chain acceptor framework sequence (for
CDR-L1, CDR-L2, and CDR-L3 sequences). In an embodiment, a human
heavy chain acceptor framework sequence of a binding protein of the
invention is selected from any of the human heavy chain acceptor
framework sequences of Table 3 and a human light chain acceptor
framework sequence of binding protein of the invention is selected
from any of the human light chain acceptor framework sequences of
Table 4. Accordingly, in an embodiment, a human acceptor framework
sequence of a binding protein according to the invention is
selected from the group consisting of SEQ ID NOS: 10-17 (which are
human heavy chain acceptor framework sequences) and SEQ ID NOS:
18-25 (which are human light chain acceptor framework sequences).
In an embodiment, the human acceptor framework sequences are
selected from the groups consisting of SEQ ID NOS: 10-13 (heavy
chain), 14-17 (heavy chain), 18-21 (light chain), and 22-25 (light
chain).
[0015] An IL-1.beta. binding protein may comprise a human acceptor
framework comprising at least one framework region (FR) amino acid
substitution, wherein the amino acid sequence of the framework is
at least 65% identical to the sequence of said human acceptor
framework and comprises at least 70 amino acid residues identical
to said human acceptor framework. In another embodiment, an
IL-1.beta. binding protein of the invention comprises a human
acceptor framework, wherein said acceptor framework comprises at
least one framework region amino acid substitution at a key
residue, said key residue selected from the group consisting
of:
a residue adjacent to a CDR; a glycosylation site residue; a rare
residue; a residue capable of interacting with human IL-1.beta.; a
residue capable of interacting with a CDR; a canonical residue; a
contact residue between heavy chain variable region and light chain
variable region; a residue within a Vernier zone; and a residue in
a region that overlaps between a Chothia-defined variable heavy
chain CDR1 and a Kabat-defined first heavy chain framework.
[0016] In an embodiment, an IL-1.beta. binding protein may comprise
a key residue, wherein said key residue is selected from the group
consisting of: 1H, 12H, 24H, 27H, 29H, 37H, 48H, 49H, 67H, 71H,
73H, 76H, 78H, 94H, 1L, 2L, 3L, 4L, 43L, 49L, 64L, 83L (all Kabat
numbering). In yet another embodiment, an IL-1.beta. binding
protein according to the invention comprises a consensus human
variable domain that is a consensus human variable domain described
herein. In another aspect, the invention provides IL-1.beta.
binding proteins comprising at least one variable domain comprising
an amino acid sequence selected from the group consisting of: 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, SEQ ID NO: 35, SEQ ID NO: 36, SEQ
ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
[0017] In another aspect, the invention provides IL-1.beta. binding
proteins comprising a variable heavy chain polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and
SEQ ID NO:33, wherein the binding proteins are capable of binding
human IL-1.beta.. In another aspect, the invention provides
IL-1.beta. binding proteins comprising a variable light chain
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, wherein the
binding proteins are capable of binding human IL-1.beta..
[0018] In yet another aspect, the invention provides binding
proteins comprising a variable heavy chain polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and
SEQ ID NO:33, and a variable light chain polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, and SEQ ID NO:40, wherein the binding proteins are
capable of binding human IL-1.beta..
[0019] In yet another aspect, the binding proteins comprise a
variable heavy chain polypeptide and a variable light chain
polypeptide selected from the group consisting of SEQ ID NO:30 and
SEQ ID NO:36; SEQ ID NO:30 and SEQ ID NO:37; SEQ ID NO:30 and SEQ
ID NO:39; and SEQ ID NO:30 and SEQ ID NO:40.
[0020] In yet another aspect, the binding proteins comprise a
variable heavy chain polypeptide and a variable light chain
polypeptide selected from the group consisting of SEQ ID NO:33 and
SEQ ID NO:36; SEQ ID NO:33 and SEQ ID NO:37; SEQ ID NO:33 and SEQ
ID NO:39; and SEQ ID NO:33 and SEQ ID NO:40.
[0021] In another aspect, the invention provide a binding protein
described above, wherein said binding protein is an immunoglobulin
molecule, a disulfide linked Fv, a monoclonal antibody, a scFv, a
chimeric antibody, a single domain antibody, a CDR-grafted
antibody, a diabody, a humanized antibody, a multispecific
antibody, an Fab, a dual specific antibody, a DVD-Ig.TM. binding
protein, a Fab', a bispecific antibody, an F(ab')2, or an Fv.
[0022] In another aspect, a binding protein described above
comprises a heavy chain immunoglobulin constant domain selected
from the group consisting of a human IgM constant domain, a human
IgG4 constant domain, a human IgG1 constant domain, a human IgE
constant domain, a human IgG2 constant domain, a human IgG3
constant domain, and a human IgA constant domain. In yet another
aspect, a binding protein of the invention further comprises a
heavy chain constant region having an amino acid sequence selected
from the group consisting of SEQ ID NO:2 and SEQ ID NO:3, and
additionally a light chain constant region having an amino acid
sequence selected from the group consisting of SEQ ID NO:4 and SEQ
ID NO:5. In another aspect of the invention, binding proteins of
the invention are capable of modulating a biological function of
human IL-1.beta. and additionally capable of neutralizing human
IL-1.beta.. In one aspect of the invention, a binding protein of
the invention has an on rate constant (Kon) to said target selected
from the group consisting of at least about
10.sup.2M.sup.-1s.sup.-1; at least about 10.sup.3M.sup.-1s.sup.-1;
at least about 10.sup.4M.sup.-1s.sup.-1; at least about
10.sup.5M.sup.-1s.sup.-1; and at least about
10.sup.6M.sup.-1s.sup.-1, as measured by surface plasmon
resonance.
[0023] In another aspect, an IL-1.beta. binding protein of the
invention has an off rate constant (Koff) to the target selected
from the group consisting of at most about 10.sup.-3 s.sup.-1; at
most about 10.sup.-4 s.sup.-1; at most about 10.sup.-5 s.sup.-1;
and at most about 10.sup.-6 s.sup.-1, as measured by surface
plasmon resonance.
[0024] In yet another aspect, an IL-1.beta. binding protein of the
invention has a dissociation constant (KD) to the IL-1.beta. target
molecule selected from the group consisting of at most about
10.sup.-7M; at most about 10.sup.-8M; at most about 10.sup.-9M; at
most about 10.sup.-10M; at most about 10.sup.-11M; at most about
10.sup.-12M; and at most about 10.sup.-13M. Additionally, the
binding proteins have a dissociation constant (KD) to IL-1.beta.
selected from the group consisting of about 1.31.times.10.sup.-10M;
about 1.47.times.10.sup.10M; m about 1.61.times.10.sup.-10M; about
1.86.times.10.sup.-10M; about 2.02.times.10.sup.-10M; about
2.06.times.10.sup.10M; m about 2.3.times.10.sup.-10M; and about
2.84.times.10.sup.-10M.
[0025] In another aspect of the invention, binding proteins of the
invention further comprise an agent selected from the group
consisting of an immunoadhesion molecule, an imaging agent, a
therapeutic agent, and a cytotoxic agent. The imaging agent can be
any imaging agent known in the art, including but not limited to, a
radiolabel (including, but not limited to, 3H, 14C, 35S, 90Y, 99Tc,
111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzyme, a
fluorescent label, a luminescent label, a bioluminescent label, a
magnetic label, or a biotin molecule. The therapeutic or cytotoxic
agent can be an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic
agent.
[0026] In another aspect, an IL-1.beta. binding protein of the
invention is glycosylated. In an embodiment, the glycosylation is a
human glycosylation pattern.
[0027] In one aspect of the invention, an IL-1.beta. binding
protein is a crystal. In one embodiment, the crystal is a
carrier-free pharmaceutical controlled release crystal. In another
embodiment, the crystallized binding protein has a greater half
life in vivo than its soluble counterpart. In still another
embodiment, the crystallized binding protein retains biological
activity after crystallization.
[0028] One aspect of the invention pertains to an isolated nucleic
acid encoding any one of the binding proteins, or antigen-binding
portion thereof, disclosed above. In an embodiment, the invention
provides an isolated nucleic acid that encodes a polypeptide
selected from the group consisting of: a polypeptide comprising a
heavy chain variable domain (VH), wherein the heavy chain variable
domain comprises a CDR-H1, a CDR-H2, and/or a CDR-H3 described
herein; a polypeptide comprising a light chain variable domain
(VL), wherein the light chain variable domain comprises a CDR-L1, a
CDR-L2, and/or a CDR-L3 described herein, or a combination of both
polypeptides.
[0029] A further embodiment of the invention provides a vector
comprising the isolated nucleic acid disclosed above wherein the
vector is selected from the group consisting of pcDNA, pTT
(Durocher et al. (2002) Nucl. Acids Res. 30(2e9):1-9), pTT3 (pTT
with additional multiple cloning site), pEFBOS (Mizushima and
Nagata (1990) Nucl. Acids Res. 18(17):5322), pBV, pJV, pBJ, and
pHybE.
[0030] In another aspect, a host cell is transformed with a vector
disclosed herein. In one embodiment, the host cell is a prokaryotic
cell including, but not limited to, Escherichia coli. In another
embodiment, the host cell is a eukaryotic cell including, but not
limited to, a protist cell, an animal cell, a plant cell, and a
fungal cell. In another embodiment, the host cell is a mammalian
cell including, but not limited to, CHO cells and COS cells, or a
fungal cell such as, for example, Saccharomyces cerevisiae, or an
insect cell such as, for example, Sf9. In another aspect, the
invention provides a method of producing a binding protein that
binds IL-1.beta., comprising culturing any one of the host cells
disclosed above in a culture medium under conditions sufficient to
produce a binding protein that binds IL-1.beta.. In another
embodiment, the invention provides a binding protein produced
according to the method disclosed herein. In an embodiment, the
invention provides a composition for the release of a binding
protein wherein the composition comprises a formulation which in
turn comprises a crystallized binding protein, crystallized
antibody construct, or crystallized antibody conjugate as disclosed
herein, an ingredient, and at least one polymeric carrier. In an
embodiment, the polymeric carrier is one or more polymers selected
from the group consisting of: poly (acrylic acid), poly
(cyanoacrylates), poly (amino acids), poly (anhydrides), poly
(depsipeptide), poly (esters), poly (lactic acid), poly
(lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly
(caprolactone), poly (dioxanone); poly (ethylene glycol), poly
((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly
(ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone),
maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives, collagen,
fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, blends and copolymers
thereof. In another aspect, the ingredient is selected from the
group consisting of albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-b-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol. In another embodiment, the invention provides
a method for treating a mammal comprising the step of administering
to the mammal an effective amount of a composition disclosed
herein.
[0031] The invention also provides pharmaceutical compositions
comprising an IL-1.beta. binding protein (or an IL-1.beta. binding
portion thereof) as disclosed herein and a pharmaceutically
acceptable carrier. Such a pharmaceutical composition of the
invention can further comprise at least one additional agent. In a
particular embodiment, a pharmaceutical composition of the
invention comprises at least one additional agent for treating a
disorder in which IL-1.beta. activity is detrimental. In another
embodiment, an additional agent is selected from the group
consisting of a therapeutic agent, an imaging agent, a cytotoxic
agent, an angiogenesis inhibitor, a kinase inhibitor, a
co-stimulation molecule blocker, an adhesion molecule blocker, an
anti-cytokine antibody, a functional fragment of an anti-cytokine
antibody, methotrexate, cyclosporin, rapamycin, FK506, a detectable
label or reporter, a TNF antagonist, an anti-rheumatic, a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteroid, an anabolic steroid, an erythropoietin, an
immunization, an immunoglobulin, an immunosuppressive agent, a
growth hormone, a hormone replacement drug, a radiopharmaceutical,
an antidepressant, an antipsychotic, a stimulant, an asthma
medication, a beta agonist, an inhaled steroid, an oral steroid, an
epinephrine or analog thereof, a cytokine, and a cytokine
antagonist.
[0032] In another aspect, the invention provides a method for
inhibiting human IL-1.beta. activity comprising contacting human
IL-1.beta. with a binding protein disclosed herein such that human
IL-1.beta. activity is inhibited. In another aspect, the invention
provides a method for inhibiting human IL-1.beta. activity in a
human subject suffering from a disorder in which IL-1.beta.
activity is detrimental, comprising administering to the human
subject a binding protein disclosed herein such that human
IL-1.beta. activity in the human subject is inhibited and treatment
is achieved.
[0033] In another aspect, the invention provides a method of
treating (e.g., curing, suppressing, ameliorating, inhibiting,
delaying, or preventing the onset of, or preventing recurrence or
relapse of) an IL-1.beta. associated disorder in a subject. In an
embodiment, the method includes administering to the subject an
IL-1.beta. binding protein, e.g., an IL-1.beta. antagonist, such as
an anti-IL-1.beta. antibody, or fragment thereof, as described
herein, in an amount sufficient to treat or prevent the IL-1.beta.
associated disorder. The IL-1.beta. antagonist can be administered
to the subject, alone or in combination with other therapeutic
modalities as described herein. In an aspect of the invention, an
IL-1.beta. binding protein, or binding portion thereof, can be
employed to detect human IL-1.beta. using any of a variety of
antibody-based immunodetection systems available in the art that
employ an antibody to detect a desired target antigen (or epitope
thereof). Such immunodetection systems include, but are not limited
to, immunoprecipitation, immunblotting (Western blot, immunodot
blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), tissue immunohistochemistry, surface plasmon resonance
(SPR), sandwich immunoassay, affinity methods (e.g., affinity
beads, affinity columns), immunocompetition assay, immunochip assay
(employing binding protein attached to a silicon chip), and
fluorescence activated cell sorting (FACS). For some
immunodetection systems, an IL-1.beta. binding protein (or binding
portion thereof) as described herein is attached to a solid
substrate using methods available in the art for attaching antibody
molecules to the same solid substrate so that the attached binding
protein retains its ability to bind human IL-1.beta. during use in
the particular immunodetection system. Such solid substrates
include, but are not limited to, a cellulose-based filter paper
(e.g., cellulose, nitrocellulose, cellulose acetate filters), a
nylon filter or membrane, a plastic surface (e.g., of a microtiter
plate or dip stick), a glass substrate (e.g., beads, slides, glass
wool), a polymeric particle (e.g., agarose, polyacrylamide), and a
silicon chip. In another aspect, the invention provides a method
for detecting the presence of IL-1.beta. in a sample in vitro
(e.g., a biological sample, such as whole blood, serum, plasma,
urine, saliva, tissue biopsy). The method can be used to diagnose a
disease or disorder, e.g., an immune cell-associated disorder. The
method includes: (i) contacting a test sample or a control sample
with an IL-1.alpha. binding protein (or binding portion thereof) as
described herein; and (ii) detecting formation of a complex between
the binding protein, or binding portion thereof, and the test
sample or the control sample, wherein a statistically significant
change in the formation of the complex in the test sample relative
to the control sample, or relative to formation of the complex in
another test sample taken at an earlier time point, is indicative
of the presence of IL-1.beta. in the sample.
[0034] In yet another aspect, the invention provides a method for
detecting the presence of IL-1.beta. in vivo (e.g., in vivo imaging
in a subject). The method is used to diagnose a disease or
disorder, e.g., an IL-1.beta.-associated disorder. The method
includes: (i) administering an IL-1.beta. binding protein, or
binding portion thereof, as described herein to a test subject or a
control subject under conditions that allow binding of the binding
protein, or binding portion thereof, to IL-1.beta.; and (ii)
detecting formation of a complex between the binding protein, or
binding portion thereof, and IL-1.beta., wherein a statistically
significant change in the formation of the complex in the test
subject relative to the control subject, or relative to the
formation of the complex in the test subject at an earlier time
point, is indicative of the presence of IL-1.beta..
[0035] In another aspect, the binding proteins of the invention are
useful for treating a disorder selected from the group consisting
of rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis,
reactive arthritis, spondyloarthropathy, systemic lupus
erythematosus, Crohn's disease, ulcerative colitis, inflammatory
bowel disease, insulin dependent diabetes mellitus, thyroiditis,
asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft
versus host disease, organ transplant rejection, acute or chronic
immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpura,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia areata, seronegative arthropathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthropathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, hepatitis B, hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasculitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, drug-induced hepatitis, non-alcoholic steatohepatitis,
allergy and asthma, group B streptococci (GB S) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Th1
Type mediated diseases, acute and chronic pain (different forms of
pain), and cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer and hematopoietic
malignancies (leukemia and lymphoma), abetalipoproteinemia,
acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti CD3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aortic and peripheral
aneurysms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chronic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic arteriosclerotic disease, diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, Epstein-Barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant
rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular
nephritis, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallervorden-Spatz disease,
Hashimoto's thyroiditis, hay fever, heart transplant rejection,
hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis A, His bundle arrhythmias, HIV infection/HIV neuropathy,
Hodgkin's disease, hyperkinetic movement disorders,
hypersensitivity reactions, hypersensitivity pneumonitis,
hypertension, hypokinetic movement disorders,
hypothalamic-pituitary-adrenal axis evaluation, idiopathic
Addison's disease, idiopathic pulmonary fibrosis, antibody mediated
cytotoxicity, asthenia, infantile spinal muscular atrophy,
inflammation of the aorta, influenza A, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis (JRA), juvenile spinal muscular atrophy, Kaposi's
sarcoma, kidney transplant rejection, legionella, leishmaniasis,
leprosy, lesions of the corticospinal system, lipedema, liver
transplant rejection, lymphedema, malaria, malignant lymphoma,
malignant histiocytosis, malignant melanoma, meningitis,
meningococcemia, metabolic/idiopathic, migraine headache,
mitochondrial multi-system disorder, mixed connective tissue
disease, monoclonal gammopathy, multiple myeloma, multiple systems
degenerations (Mencel Dejerine-Thomas Shi-Drager and
Machado-Joseph), myasthenia gravis, mycobacterium avium
intracellulare, mycobacterium tuberculosis, myelodysplastic
syndrome, myocardial infarction, myocardial ischemic disorders,
nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis,
nephrosis, neurodegenerative diseases, neurogenic I muscular
atrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of
the abdominal aorta and its branches, occlusive arterial disorders,
OKT3.RTM. therapy, orchitis/epidydimitis, orchitis/vasectomy
reversal procedures, organomegaly, osteoporosis, pancreas
transplant rejection, pancreatic carcinoma, paraneoplastic
syndrome/hypercalcemia of malignancy, parathyroid transplant
rejection, pelvic inflammatory disease, perennial rhinitis,
pericardial disease, peripheral atherosclerotic disease, peripheral
vascular disorders, peritonitis, pernicious anemia, pneumocystis
carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, and skin
changes syndrome), post perfusion syndrome, post pump syndrome,
post-MI cardiotomy syndrome, preeclampsia, progressive supranucleo
palsy, primary pulmonary hypertension, radiation therapy, Raynaud's
phenomenon, Raynaud's disease, Refsum's disease, regular narrow QRS
tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea,
senile dementia of Lewy body type, seronegative arthropathies,
shock, sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrhythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
subacute sclerosing panencephalitis, syncope, syphilis of the
cardiovascular system, systemic anaphylaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, viral
encephalitis/aseptic meningitis, viral-associated hemaphagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, acute coronary syndromes, acute
idiopathic polyneuritis, acute inflammatory demyelinating
polyradiculoneuropathy, acute ischemia, adult Still's disease,
alopecia areata, anaphylaxis, anti-phospholipid antibody syndrome,
aplastic anemia, arteriosclerosis, atopic eczema, atopic
dermatitis, autoimmune dermatitis, autoimmune disorder associated
with streptococcus infection, autoimmune enteropathy, autoimmune
hearing loss, autoimmune lymphoproliferative syndrome (ALPS),
autoimmune myocarditis, autoimmune premature ovarian failure,
blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular
disease, catastrophic antiphospholipid syndrome, celiac disease,
cervical spondylosis, chronic ischemia, cicatricial pemphigoid,
clinically isolated syndrome (CIS) with risk for multiple
sclerosis, conjunctivitis, childhood onset psychiatric disorder,
chronic obstructive pulmonary disease (COPD), dacryocystitis,
dermatomyositis, diabetic retinopathy, diabetes mellitus, disk
herniation, disk prolapse, drug induced immune hemolytic anemia,
endocarditis, endometriosis, endophthalmitis, episcleritis,
erythema multiforme, erythema multiforme major, gestational
pemphigoid, Guillain-Barre syndrome (GBS), hay fever, Hughes
syndrome, idiopathic Parkinson's disease, idiopathic interstitial
pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion
body myositis, infectious ocular inflammatory disease, inflammatory
demyelinating disease, inflammatory heart disease, inflammatory
kidney disease, IPF/UIP, iritis, keratitis, keratojunctivitis
sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's
paralysis, Langerhan's cell histiocytosis, livedo reticularis,
macular degeneration, microscopic polyangiitis, morbus bechterev,
motor neuron disorders, mucous membrane pemphigoid, multiple organ
failure, myasthenia gravis, myelodysplastic syndrome, myocarditis,
nerve root disorders, neuropathy, non-A non-B hepatitis, optic
neuritis, osteolysis, pauciarticular JRA, peripheral artery
occlusive disease (PAOD), peripheral vascular disease (PVD),
peripheral artery disease (PAD), phlebitis, polyarteritis nodosa
(or periarteritis nodosa), polychondritis, polymyalgia rheumatica,
poliosis, polyarticular JRA, polyendocrine deficiency syndrome,
polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome,
primary Parkinsonism, prostatitis, pure red cell aplasia, primary
adrenal insufficiency, recurrent neuromyelitis optica, restenosis,
rheumatic heart disease, SAPHO (synovitis, acne, pustulosis,
hyperostosis, and osteitis), scleroderma, secondary amyloidosis,
shock lung, scleritis, sciatica, secondary adrenal insufficiency,
silicone associated connective tissue disease, Sneddon-Wilkinson
dermatosis, spondylitis ankylosans, Stevens-Johnson syndrome (SJS),
systemic inflammatory response syndrome, temporal arteritis,
toxoplasmic retinitis, toxic epidermal necrolysis, transverse
myelitis, TRAPS (Tumor-necrosis factor receptor type 1
(TNFR)-Associated Periodic Syndrome); type B insulin resistance
with acanthosis nigricans; type 1 allergic reaction, type II
diabetes, urticaria, usual interstitial pneumonia (UIP),
vasculitis, vernal conjunctivitis, viral retinitis,
Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular
degeneration, and wound healing.
[0036] In one aspect, the binding proteins of the invention are
used to treat rheumatoid arthritis, osteoarthritis, Crohn's
disease, multiple sclerosis, insulin dependent diabetes mellitus
and psoriasis. In another aspect, the binding proteins of the
invention are also used to treat humans suffering from autoimmune
diseases, in particular those associated with inflammation,
including, ankylosing spondylitis, allergy, autoimmune diabetes,
autoimmune uveitis.
[0037] In another aspect the invention provides a method of
treating a patient suffering from a disorder in which human
IL-1.beta. is detrimental comprising the step of administering any
one of the binding proteins described herein before, concurrent
with, or after the administration of a second agent, as discussed
above. In a another embodiment the additional therapeutic agent
that can be coadministered and/or coformulated with one or more
IL-1.beta. antagonists, (e.g., anti-IL-1.beta. antibodies or
fragments thereof) includes, but is not limited to, TNF
antagonists; a soluble fragment of a TNF receptor; ENBREL.RTM.
(etanercept); TNF enzyme antagonists; TNF converting enzyme (TACE)
inhibitors; muscarinic receptor antagonists; TGF-beta antagonists;
interferon gamma; perfenidone; chemotherapeutic agents,
methotrexate; leflunomide; sirolimus (rapamycin) or an analog
thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs;
immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors;
budenoside; epidermal growth factor; corticosteroids; cyclosporine;
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor
antagonists; anti-IL-1.beta. antibodies; anti-IL-6 antibodies;
growth factors; elastase inhibitors; pyridinyl-imidazole compounds;
antibodies or agonists of TNF, LT, IL-1.alpha., IL-1.beta., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,
IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,
IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2,
CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their
ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen;
prednisolone; phosphodiesterase inhibitors; adensosine agonists;
antithrombotic agents; complement inhibitors; adrenergic agents;
IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1.beta.
converting enzyme inhibitors; TNF.alpha. converting enzyme
inhibitors; T-cell signaling inhibitors; metalloproteinase
inhibitors; 6-mercaptopurines; angiotensin converting enzyme
inhibitors; soluble cytokine receptors; soluble p55 TNF receptor;
soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R;
anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGF.beta..
[0038] In one embodiment, the pharmaceutical compositions disclosed
herein are administered to a subject by at least one mode selected
from the group consisting of parenteral, subcutaneous,
intramuscular, intravenous, intra-articular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, and transdermal routes.
[0039] One aspect of the invention provides at least one IL-1.beta.
anti-idiotypic antibody to at least one IL-1.beta. binding protein
of the invention. The anti-idiotypic antibody includes any protein
or peptide containing molecule that comprises at least a portion of
an immunoglobulin molecule such as, but not limited to, at least
one complementarily determining region (CDR) of a heavy or light
chain, or a ligand binding portion thereof, a heavy chain or light
chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that can be
incorporated into a binding protein of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0040] This invention pertains to IL-1.beta. binding proteins,
particularly anti-IL-1.beta. antibodies, or antigen-binding
portions thereof, that bind human IL-1.beta.. 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 IL-1.beta. binding portions thereof. Methods of
using the binding proteins of the invention to detect human
IL-1.beta., to inhibit human IL-1.beta. activity, either in vitro
or in vivo; and to regulate gene expression are also encompassed by
the invention.
[0041] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the term "or" includes "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0042] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics, protein and nucleic acid
chemistry and nucleic acid hybridization described herein are those
well known and commonly used in the art. The methods and techniques
of the present invention are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification unless otherwise
indicated. Enzymatic reactions and purification techniques are
performed according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0043] That the present invention may be more readily understood,
select terms are defined below.
[0044] The term "polypeptide" means any polymeric chain of amino
acids. The terms "peptide" and "protein" are used interchangeably
with the term polypeptide and also refer to a polymeric chain of
amino acids. The term "polypeptide" encompasses native or
artificial proteins, protein fragments and polypeptide analogs of a
protein sequence. A polypeptide may be monomeric or polymeric.
[0045] The term "isolated protein" or "isolated polypeptide" means
a protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state, is substantially free of
other proteins from the same species, is expressed by a cell from a
different species, or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0046] The term "recovering" means the process of rendering a
chemical species such as a polypeptide substantially free of
naturally associated components by isolation, e.g., using protein
purification techniques well known in the art.
[0047] The term "human IL-1.beta." (abbreviated herein as
hIL-1.beta., or IL-1.beta.) includes a pleiotropic cytokine
involved in various immune responses, inflammatory processes, and
hematopoiesis. The term human IL-1.beta. includes recombinant human
IL-1.beta. (rh IL-1.beta.) that can be prepared by standard
recombinant expression methods.
TABLE-US-00001 TABLE 1 Sequence of Human IL-1.beta. Sequence Amino
Acid Sequence Protein Identifier 123456789012345678901234567890
Human IL SEQ ID APVRSLNCTLRDSQQKSLVMSGPYELKALH 1.beta. NO.: 1
LQGQDMEQQVVFSMSFVQGEESNDKIPVAL GLKEKNLYLSCVLKDDKPTLQLESVDPKNY
PKKKMEKRFVFNKIEINNKLEFESAQFPNW YISTSQAENMPVFLGGTKGGQDITDFTMQF
VSS
[0048] "Biological activity" refers to all inherent biological
properties of IL-1.beta.. Biological properties of IL-1.beta.
include but are not limited to binding IL-1 receptor; (other
examples include: induction of fever, slow wave sleep, and
neutrophilia, T- and B-lymphocyte activation, fibroblast
proliferation, cytotoxicity for certain cells, induction of
collagenases, synthesis of hepatic acute phase proteins, and
increased production of colony stimulating factors and
collagen).
[0049] The terms "specific binding" or "specifically binding" in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, mean that the interaction
is dependent upon the presence of a particular structure (e.g., an
antigenic determinant or epitope) on the chemical species, for
example, an antibody recognizes and binds to a specific protein
structure rather than to proteins generally. If an antibody is
specific for epitope "A", the presence of a molecule containing
epitope A (or free, unlabeled A), in a reaction containing labeled
"A" and the antibody, will reduce the amount of labeled A bound to
the antibody.
[0050] The term "antibody" broadly refers to any immunoglobulin
(Ig) molecule comprised of four polypeptide chains, two heavy (H)
chains and two light (L) chains, or any functional fragment,
mutant, variant, or derivative thereof, that retains the essential
epitope binding features of an Ig molecule. Such mutant, variant,
or derivative antibody formats are known in the art, nonlimiting
embodiments of which are discussed below.
[0051] In a full-length antibody, 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. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[0052] The term "antigen-binding portion" of an antibody refers to
one or more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., hIL-1.alpha.). The
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also have bispecific, dual specific, or multi-specific formats;
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) an Fab fragment, which is a
monovalent fragment consisting of the VL, VH, CL and CH1 domains;
(ii) an F(ab').sub.2 fragment, which is 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) an 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, PCT Publication No. WO 90/05144, which
comprises a single variable 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 (scFvs)
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, et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123). Such
antibody binding portions are known in the art (Kontermann and
Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York.
790 pp. (ISBN 3-540-41354-5)).
[0053] The term "antibody construct" refers to a polypeptide
comprising one or more the antigen binding portions of the
invention linked to a linker polypeptide or an immunoglobulin
constant domain. Linker polypeptides comprise two or more amino
acid residues joined by peptide bonds and are used to link one or
more antigen binding portions. Such linker polypeptides are well
known in the art (see e.g., Holliger et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure
2:1121-1123). An immunoglobulin constant domain refers to a heavy
or light chain constant domain. Human IgG heavy chain (gamma) and
light chain (kappa and delta) constant domain amino acid sequences
are known in the art and represented in Table 2.
TABLE-US-00002 TABLE 2 Sequences Of Human IgG Heavy And Light Chain
Constant Domains Sequence Sequence Protein Identifier
123456789012345678901234567890 Ig gamma-1 SEQ ID NO.: 2
ASTKGPSVFFLAPSSKSTSGGTAALGCLVK constant region
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPESCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKENNYVDGVEVHNAETKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGKYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig
gamma-1 SEQ ID NO.: 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVK constant
region DYFPEPVTVSWNSGALTSGVHTFPAVLQSS mutant
GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappa constant SEQ ID NO.: 4
TVAAPSVFIFPPSDEQLKSGTASVVCLLNN region
FYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC Ig Lambda SEQ ID NO.: 5
QPKAAPSVTLFPPSSEELQANKATLVCLIS constant region
DFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTH
EGSTVEKTVAPTECS
[0054] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecule, formed by
covalent or noncovalent association of the antibody or antigen
binding portion thereof 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 et al. (1995) Human Antibod. 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 et al. (1994) Mol. Immunol. 31:1047-1058). Antigen
binding portions of antibodies, 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, antigen binding portions
thereof, and immunoadhesion molecules can be obtained using
standard recombinant DNA techniques, as described herein.
[0055] An "isolated antibody" refers to an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
hIL-1.beta. is substantially free of antibodies that specifically
bind antigens other than hIL-1.beta.). An isolated antibody that
specifically binds hIL-1.beta. may, however, have cross-reactivity
to other antigens, such as IL-1.beta. molecules from other species.
Moreover, an isolated antibody may be substantially free of other
cellular material and/or chemicals.
[0056] The term "human antibody" is intended to include antibodies
having variable and constant regions derived from human germline
immunoglobulin sequences. The human antibodies of the invention may
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo),
for example in the CDRs and in particular CDR3. However, the term
"human antibody" does not 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.
[0057] The term "recombinant human antibody" includes 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 C, below), antibodies isolated from a recombinant,
combinatorial human antibody library (Hoogenboom (1997) Trends
Biotechnol. 15:62-70; Azzazy and Highsmith (2002) Clin. Biochem.
35:425-445; Gavilondo and Larrick (2000) BioTechniques 29:128-145;
Hoogenboom and Chames (2000) Immunology Today 21:371-378),
antibodies isolated from an animal (e.g., a mouse) that is
transgenic for human immunoglobulin genes (see, e.g., Taylor et al.
(1992) Nucl. Acids Res. 20:6287-6295; Kellermann and Green (2002)
Curr. Opin. Biotechnol. 13:593-597; Little et al. (2000) Immunol.
Today 21:364-370) 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.
[0058] The term "chimeric antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0059] The term "CDR-grafted antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL regions are replaced with CDR sequences of
another species, such as antibodies that have human heavy and light
chain variable regions in which one or more of the human CDRs
(e.g., CDR3) has been replaced with murine CDR sequences, for
example, as obtained from a murine monoclonal antibody to human
IL-1.beta..
[0060] The term "CDR" refers to the complementarity determining
region within antibody variable sequences. There are three CDRs in
each of the variable regions of the heavy chain and the light
chain, which are designated CDR1, CDR2, and CDR3, for each of the
variable regions. The term "CDR set" refers to a group of three
CDRs that occur in a single variable region (i.e., VH or VL) of an
antigen binding site. The exact boundaries of these CDRs have been
defined differently according to different systems. The system
described by Kabat (Kabat et al. (1987, 1991) Sequences of Proteins
of Immunological Interest (National Institutes of Health, Bethesda,
Md.) not only provides an unambiguous residue numbering system
applicable to any variable region of an antibody, but also provides
precise residue boundaries defining the three CDRs. These CDRs may
be referred to as Kabat CDRs. Chothia and coworkers (Chothia and
Lesk (1987) J. Mol. Biol. 196:901-917 and Chothia et al. (1989)
Nature 342:877-883) found that certain sub-portions within Kabat
CDRs adopt nearly identical peptide backbone conformations, despite
having great diversity at the level of amino acid sequence. These
sub-portions were designated as L1, L2, and L3 or H1, H2, and H3,
where the "L" and the "H" designates the light chain and the heavy
chains regions, respectively. These regions may be referred to as
Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
Other boundaries defining CDRs overlapping with the Kabat CDRs have
been described by Padlan et al. (1995) FASEB J. 9:133-139 and
MacCallum (1996) J. Mol. Biol. 262(5):732-745). Still other CDR
boundary definitions may not strictly follow one of the above
systems, but will nonetheless overlap with the Kabat CDRs, although
they may be shortened or lengthened in light of prediction or
experimental findings that particular residues or groups of
residues or even entire CDRs do not significantly impact antigen
binding. The methods used herein may utilize CDRs defined according
to any of these systems, although certain embodiments use Kabat or
Chothia defined CDRs.
[0061] The terms "Kabat numbering", "Kabat definition and "Kabat
labeling" are used interchangeably herein. These terms refer to a
system of numbering amino acid residues which are more variable
(i.e., hypervariable) than other amino acid residues in the heavy
and light chain variable regions of an antibody, or an antigen
binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.
190:382-391 and Kabat et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242). For the heavy
chain variable region, the hypervariable region ranges from amino
acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for
CDR2, and amino acid positions 95 to 102 for CDR3. For the light
chain variable region, the hypervariable region ranges from amino
acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for
CDR2, and amino acid positions 89 to 97 for CDR3.
[0062] The growth and analysis of extensive public databases of
amino acid sequences of variable heavy and light regions over the
past twenty years have led to the understanding of the typical
boundaries between framework regions (FR) and CDR sequences within
variable region sequences and enabled persons skilled in this art
to accurately determine the CDRs according to Kabat numbering,
Chothia numbering, or other systems. See, e.g., Martin, In
Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag,
Berlin, 2001), chapter 31, pages 432-433. A useful method of
determining the amino acid sequences of Kabat CDRs within the amino
acid sequences of variable heavy (VH) and variable light (VL)
regions is provided below:
[0063] To identify a CDR-L1 amino acid sequence:
[0064] Starts approximately 24 amino acid residues from the amino
terminus of the VL region;
[0065] Residue before the CDR-L1 sequence is always cysteine
(C);
[0066] Residue after the CDR-L1 sequence is always a tryptophan (W)
residue, typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln
(W-L-Q), Trp-Phe-Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L);
[0067] Length is typically 10 to 17 amino acid residues.
[0068] To identify a CDR-L2 amino acid sequence:
[0069] Starts always 16 residues after the end of CDR-L1;
[0070] Residues before the CDR-L2 sequence are generally Ile-Tyr
(I-Y), but also Val-Tyr (V-Y), Ile-Lys (I-K), and Ile-Phe
(I-F);
[0071] Length is always 7 amino acid residues.
[0072] To identify a CDR-L3 amino acid sequence:
[0073] Starts always 33 amino acids after the end of CDR-L2;
[0074] Residue before the CDR-L3 amino acid sequence is always a
cysteine (C);
[0075] Residues after the CDR-L3 sequence are always Phe-Gly-X-Gly
(F-G-X-G) (SEQ ID NO:6), where X is any amino acid;
[0076] Length is typically 7 to 11 amino acid residues.
[0077] To identify a CDR-H1 amino acid sequence:
[0078] Starts approximately 31 amino acid residues from amino
terminus of VH region and always 9 residues after a cysteine
(C);
[0079] Residues before the CDR-H1 sequence are always
Cys-X-X-X-X-X-X-X-X (SEQ ID NO:7), where X is any amino acid;
[0080] Residue after CDR-H1 sequence is always a Trp (W), typically
Trp-Val (W-V), but also Trp-Ile (W-I), and Trp-Ala (W-A);
[0081] Length is typically 5 to 7 amino acid residues.
[0082] To identify a CDR-H2 amino acid sequence:
[0083] Starts always 15 amino acid residues after the end of
CDR-H1;
[0084] Residues before CDR-H2 sequence are typically
Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ ID NO:8), but other variations
also;
[0085] Residues after CDR-H2 sequence are
Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala
(K/R-L/I/V/F/T/A-T/S/I/A);
[0086] Length is typically 16 to 19 amino acid residues.
[0087] To identify a CDR-H3 amino acid sequence:
[0088] Starts always 33 amino acid residues after the end of CDR-H2
and always 3 after a cysteine (C)'
[0089] Residues before the CDR-H3 sequence are always Cys-X-X
(C-X-X), where X is any amino acid, typically Cys-Ala-Arg
(C-A-R);
[0090] Residues after the CDR-H3 sequence are always Trp-Gly-X-Gly
(W-G-X-G) (SEQ ID NO:9), where X is any amino acid;
[0091] Length is typically 3 to 25 amino acid residues.
[0092] The terms "acceptor" and "acceptor antibody" refer to the
antibody or nucleic acid sequence providing or encoding at least
80%, at least 85%, at least 90%, at least 95%, at least 98% or 100%
of the amino acid sequences of one or more of the framework
regions. In some embodiments, the term "acceptor" refers to the
antibody amino acid or nucleic acid sequence providing or encoding
the constant region(s). In yet another embodiment, the term
"acceptor" refers to the antibody amino acid or nucleic acid
sequence providing or encoding one or more of the framework regions
and the constant region(s). In a specific embodiment, the term
"acceptor" refers to a human antibody amino acid or nucleic acid
sequence that provides or encodes at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, or 100% of the amino acid
sequences of one or more of the framework regions. In accordance
with this embodiment, an acceptor may contain at least 1, at least
2, at least 3, least 4, at least 5, or at least 10 amino acid
residues that does (do) not occur at one or more specific positions
of a human antibody. An acceptor framework region and/or acceptor
constant region(s) may be, e.g., derived or obtained from a
germline antibody gene, a mature antibody gene, a functional
antibody (e.g., antibodies well-known in the art, antibodies in
development, or antibodies commercially available).
[0093] The term "canonical" residue refers to a residue in a CDR or
framework that defines a particular canonical CDR structure as
defined by Chothia et al. (1987) J. Mol. Biol. 196:901-917; Chothia
et al. (1992) J. Mol. Biol. 227:799. According to Chothia et al.,
critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0094] The terms "donor" and "donor antibody" refer to an antibody
providing one or more CDRs. In an embodiment, the donor antibody is
an antibody from a species different from the antibody from which
the framework regions are obtained or derived. In the context of a
humanized antibody, the term "donor antibody" refers to a non-human
antibody providing one or more CDRs.
[0095] The term "framework" or "framework sequence" refers to the
remaining sequences of a variable region minus the CDRs. Because
the exact definition of a CDR sequence can be determined by
different systems, the meaning of a framework sequence is subject
to correspondingly different interpretations. The six CDRs (CDR-L1,
-L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy
chain) also divide the framework regions on the light chain and the
heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each
chain, in which CDR1 is positioned between FR1 and FR2, CDR2
between FR2 and FR3, and CDR3 between FR3 and FR4. Without
specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a
framework region, as referred by others, represents the combined
FR's within the variable region of a single, naturally occurring
immunoglobulin chain. A FR represents one of the four sub-regions,
and FRs represents two or more of the four sub-regions constituting
a framework region.
[0096] Human heavy chain and light chain acceptor sequences are
known in the art. In one embodiment of the invention the human
heavy chain and light chain acceptor sequences are selected from
the sequences described in Table 3 and Table 4.
TABLE-US-00003 TABLE 3 Heavy Chain Acceptor Sequences SEQ ID
Protein Sequence No. region 12345678901234567890123456789012 10
VH4-59 FR1 QVQLQESGPGLVKPSETLSLTCTVSGGSIS 11 VH4-59 FR2
WIRQPPGKGLEWIG 12 VH4-59 FR3 RVTISVDTSKNQFSLKLSSVTAADT AVYYCAR 13
JH4 FR4 WGQGTLVTVSS 14 VH3-53 FR1 EVQLVESGGGLIQPGGSLRLSCAASGFTVS 15
VH3-53 FR2 WVRQAPGKGLEWVS 16 VH3-53 FR3 RFTISRDNSKNTLYLQMNSLRAEDT
AVYYCAR 17 hJH4 FR4 WGQGTLVTVSS
TABLE-US-00004 TABLE 4 Light Chain Acceptor Sequences SEQ ID
Protein Sequence No. region 12345678901234567890123456789012 18 018
FR1 DIQMTQSPSSLSASVGDRVTITC 19 018 FR2 WYQQKPGKAPKLLIY 20 018 FR3
GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 21 JK2 FR4 FGQGTKLEIK 22 A14 FR1
DVVMTQSPAFLSVTPGEKVTITC 23 A14 FR2 WYQQKPDQAPKLLIK 24 A14 FR3
GVPSRFSGSGSGTDFTFTISSLEAEDAATYYC 25 JK2 FR4 FGQGTKLEIK
[0097] The term "germline antibody gene" or "gene fragment" refers
to an immunoglobulin sequence encoded by non-lymphoid cells that
have not undergone the maturation process that leads to genetic
rearrangement and mutation for expression of a particular
immunoglobulin. (see, e.g., Shapiro et al. (2002) Crit. Rev.
Immunol. 22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med.
Biol. 484:13-30). One of the advantages provided by various
embodiments of the present invention stems from the recognition
that germline antibody genes are more likely than mature antibody
genes to conserve essential amino acid sequence structures
characteristic of individuals in the species, hence less likely to
be recognized as from a foreign source when used therapeutically in
that species.
[0098] The term "key" residues refer to certain residues within the
variable region that have more impact on the binding specificity
and/or affinity of an antibody, in particular a humanized antibody.
A key residue includes, but is not limited to, one or more of the
following: a residue that is adjacent to a CDR, a potential
glycosylation site (can be either N- or O-glycosylation site), a
rare residue, a residue capable of interacting with the antigen, a
residue capable of interacting with a CDR, a canonical residue, a
contact residue between heavy chain variable region and light chain
variable region, a residue within the Vernier zone, and a residue
in the region that overlaps between the Chothia definition of a
variable heavy chain CDR1 and the Kabat definition of the first
heavy chain framework.
[0099] The term "humanized antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which non-human CDR sequences are introduced into
human VH and VL sequences to replace the corresponding non-human
framework (FR) sequences. For example, a "humanized antibody" is an
antibody or a variant, derivative, analog or fragment thereof which
immunospecifically binds to an antigen of interest and which
comprises a framework (FR) region having substantially the amino
acid sequence of a human antibody and a complementary determining
region (CDR) having substantially the amino acid sequence of a
non-human antibody. The term "substantially" in the context of a
CDR refers to a CDR having an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 95%, at least 98% or at least 99%
identical to the amino acid sequence of a non-human antibody CDR. A
humanized antibody comprises substantially all of at least one, and
typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in
which all or substantially all of the CDR regions correspond to
those of a non-human immunoglobulin (i.e., donor antibody) and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. In an embodiment, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. In
some embodiments, a humanized antibody contains both the light
chain as well as at least the variable domain of a heavy chain. The
antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions
of the heavy chain. In some embodiments, a humanized antibody only
contains a humanized light chain. In some embodiments, a humanized
antibody only contains a humanized heavy chain. In specific
embodiments, a humanized antibody only contains a humanized
variable domain of a light chain and/or humanized heavy chain.
[0100] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well-known
in the art.
[0101] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In an embodiment, such mutations, however, will not be extensive.
Usually, at least 80%, at least 85%, at least 90%, and at least 95%
of the humanized antibody residues will correspond to those of the
parental FR and CDR sequences. The term "consensus framework"
refers to the framework region in the consensus immunoglobulin
sequence. The term "consensus immunoglobulin sequence" refers to
the sequence formed from the most frequently occurring amino acids
(or nucleotides) in a family of related immunoglobulin sequences
(See, e.g., Winnaker (1987) From Genes to Clones
(Verlagsgesellschaft, Weinheim, Germany). A "consensus
immunoglobulin sequence" can thus comprise a "consensus variable
domain" and/or a "consensus constant domain". A "consensus variable
domain" can in turn comprise one or more "consensus framework
regions" and/or one or more "consensus CDRs". In a family of
immunoglobulins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0102] The term "Vernier" zone refers to a subset of framework
residues that may adjust CDR structure and fine-tune the fit to
antigen as described by Foote and Winter (1992) J. Mol. Biol.
224:487-499). Vernier zone residues form a layer underlying the
CDRs and may impact on the structure of CDRs and the affinity of
the antibody.
[0103] The term "multivalent binding protein" is used in this
specification to denote a binding protein comprising two or more
antigen binding sites. The multivalent binding protein is
engineered to have the three or more antigen binding sites, and is
generally not a naturally occurring antibody. The term
"multispecific binding protein" refers to a binding protein capable
of binding two or more related or unrelated targets. Dual variable
domain (DVD) binding proteins, are binding proteins that comprise
two or more antigen binding sites and are tetravalent or
multivalent binding proteins. Such DVD binding proteins may be
monospecific, i.e., capable of binding one antigen or
multispecific, i.e., capable of binding two or more antigens. DVD
binding proteins comprising two heavy chain DVD polypeptides and
two light chain DVD polypeptides are referred to as DVD-Ig.TM.
molecule. Each half of a DVD-Ig molecule comprises a heavy chain
DVD-Ig polypeptide, and a light chain DVD polypeptide, and two
antigen binding sites. Each binding site comprises a heavy chain
variable domain and a light chain variable domain with a total of 6
CDRs involved in antigen binding per antigen binding site. DVD
binding proteins and methods of making DVD binding proteins are
disclosed in U.S. Pat. No. 7,612,181, which is incorporated herein
by reference.
[0104] One aspect of the invention pertains to a DVD binding
protein comprising binding proteins capable of binding human
IL-1.beta.. In another aspect, the DVD binding protein is capable
of binding IL-1.beta. and a second target. In one embodiment, the
DVD binding protein is capable of binding IL-1.alpha., and
IL-1.beta..
[0105] The term "neutralizing" refers to neutralization of
biological activity of a cytokine when a binding protein
specifically binds the cytokine. In an embodiment, a neutralizing
binding protein is a neutralizing antibody whose binding to
hIL-1.beta. results in inhibition of a biological activity of
hIL-1.beta.. Preferably the neutralizing binding protein binds
hIL-1.beta. and reduces a biologically activity of hIL-1.beta. by
at least about 20%, at least about 40%, at least about 60%, at
least about 80%, at least about 85%, at least about 85%, at least
about 90%, at least about 95%, or at least about 100%. Inhibition
of a biological activity of hIL-1.beta. by a neutralizing binding
protein can be assessed by measuring one or more indicators of
hIL-1.beta. biological activity well known in the art. The term
"epitope" includes any polypeptide determinant capable of specific
binding to an immunoglobulin or T-cell receptor. In certain
embodiments, epitope determinants include chemically active surface
groupings of molecules such as amino acids, sugar side chains,
phosphoryl, or sulfonyl, and, in certain embodiments, may have
specific three dimensional structural characteristics, and/or
specific charge characteristics. An epitope is a region of an
antigen that is bound by an antibody. An epitope thus consists of
the amino acid residues of a region of an antigen (or fragment
thereof) known to bind to the complementary site on the specific
binding partner. An antigenic fragment can contain more than one
epitope. In certain embodiments, an antibody is said to
specifically bind an antigen when it recognizes its target antigen
in a complex mixture of proteins and/or macromolecules. Antibodies
are said to "bind to the same epitope" if the antibodies
cross-compete (one prevents the binding or modulating effect of the
other). In addition structural definitions of epitopes
(overlapping, similar, identical) are informative, but functional
definitions are often more relevant as they encompass structural
(binding) and functional (modulation, competition) parameters.
[0106] The term "surface plasmon resonance" 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 BIACOREa system
(Biacore International AB, a GE Healthcare company, Uppsala, Sweden
and Piscataway, N.J.). For further descriptions, see Jonsson, U. et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al. (1991)
BioTtechniques 11:620-627; Johnsson, B. et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnsson, B. et al. (1991) Anal. Biochem.
198:268-277.
[0107] The term "Kon" refers to the on rate constant for
association of a binding protein (e.g., an antibody) to the antigen
to form the, e.g., antibody/antigen complex as is known in the art.
The "Kon" also is known by the terms "association rate constant,"
or "ka," as used interchangeably herein. This value indicating the
binding rate of an antibody to its target antigen or the rate of
complex formation between an antibody and antigen also is shown by
the equation:
Antibody ("Ab")+Antigen ("Ag").fwdarw.Ab-Ag.
[0108] The term "Koff" refers to the off rate constant for
dissociation of a binding protein (e.g., an antibody) from the,
e.g., antibody/antigen complex as is known in the art. The "Koff"
also is known by the terms "dissociation rate constant" or "kd" as
used interchangeably herein. This value indicates the dissociation
rate of an antibody from its target antigen or separation of Ab-Ag
complex over time into free antibody and antigen as shown by the
equation below:
Ab+Ag.rarw.Ab-Ag
[0109] The terms "equilibrium dissociation constant" or "KD," as
used interchangeably herein, refer to the value obtained in a
titration measurement at equilibrium, or by dividing the
dissociation rate constant (koff) by the association rate constant
(kon). The association rate constant, the dissociation rate
constant, and the equilibrium dissociation constant are used to
represent the binding affinity of an antibody to an antigen.
Methods for determining association and dissociation rate constants
are well known in the art. Using fluorescence-based techniques
offers high sensitivity and the ability to examine samples in
physiological buffers at equilibrium. Other experimental approaches
and instruments such as a BIACOREa (biomolecular interaction
analysis) assay can be used (e.g., instrument available from
Biacore International AB, a GE Healthcare company, Uppsala,
Sweden). Additionally, a KinExA.RTM. (Kinetic Exclusion Assay)
assay, available from Sapidyne Instruments (Boise, Id.) can also be
used.
[0110] The term "labeled binding protein" refers to a protein with
a label incorporated that provides for the identification of the
binding protein. In one aspect, the label is a detectable marker,
e.g., incorporation of a radiolabeled amino acid or attachment to a
polypeptide of biotinyl moieties that can be detected by marked
avidin (e.g., streptavidin containing a fluorescent marker or
enzymatic activity that can be detected by optical or colorimetric
methods). Examples of labels for polypeptides include, but are not
limited to, the following: radioisotopes or radionuclides (e.g.,
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho and .sup.153Sm);
fluorescent labels (e.g., FITC, rhodamine, and lanthanide
phosphors), enzymatic labels (e.g., horseradish peroxidase,
luciferase, alkaline phosphatase); chemiluminescent markers;
biotinyl groups; predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, and epitope
tags); and magnetic agents, such as gadolinium chelates.
[0111] The term "antibody conjugate" refers to a binding protein,
such as an antibody, chemically linked to a second chemical moiety,
such as a therapeutic or cytotoxic agent. The term "agent" denotes
a chemical compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials. In one
aspect, the therapeutic or cytotoxic agents include, but are not
limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
[0112] The terms "crystal", and "crystallized" refer to an
antibody, or antigen binding portion thereof, that exists in the
form of a crystal. Crystals are one form of the solid state of
matter, which is distinct from other forms such as the amorphous
solid state or the liquid crystalline state. Crystals are composed
of regular, repeating, three-dimensional arrays of atoms, ions,
molecules (e.g., proteins such as antibodies), or molecular
assemblies (e.g., antigen/antibody complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships that are well-understood in the field.
The fundamental unit, or building block, that is repeated in a
crystal is called the asymmetric unit. Repetition of the asymmetric
unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the crystal.
Repetition of the unit cell by regular translations in all three
dimensions provides the crystal. See Giege and Ducruix (1999)
Chapter 1, In Crystallization of Nucleic Acids and Proteins, a
Practical Approach, 2nd ed., (Ducruix and Giege, eds.) (Oxford
University Press, New York, 1999) pp. 1-16.
[0113] The term "polynucleotide" means a polymeric form of two or
more nucleotides, either ribonucleotides or deoxynucleotides or a
modified form of either type of nucleotide. The term includes
single and double stranded forms of DNA or RNA but in an embodiment
is double-stranded DNA.
[0114] The term "isolated polynucleotide" means a polynucleotide
(e.g., of genomic, cDNA, or synthetic origin, or a combination
thereof) that is not associated with all or a portion of a
polynucleotide with which it is associated in nature, with which it
is operably linked to in nature, or with which it occurs in nature
as part of a larger sequence.
[0115] The term "vector" refers 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.
[0116] The term "operably linked" refers to a positioning of
components such that they function in their intended manner. A
control sequence "operably linked" to a coding sequence is ligated
in such a way that expression of the coding sequence is achieved
under conditions compatible with the control sequences. "Operably
linked" sequences include expression control sequences that are
contiguous with a nucleic acid of interest, expression control
sequences that act in trans i.e., are located on a different
nucleic acid molecule than a nucleic acid of interest but
nevertheless exert control over the nucleic acid of interest, and
expression control sequences that are located on the same nucleic
acid molecule as, but at a distance from, a nucleic acid of
interest. The term "expression control sequence" refers to
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0117] "Transformation" to any process by which exogenous DNA
enters a host cell. Transformation may occur under natural or
artificial conditions using various methods well known in the art
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell, for example. The method is
selected based on the host cell being transformed and may include,
but is not limited to, viral infection, electroporation,
lipofection, and particle bombardment. Such "transformed" cells
include stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome. They also include cells
which transiently express the inserted DNA or RNA for limited
periods of time.
[0118] The term "recombinant host cell" (or simply "host cell")
refers to a cell into which exogenous DNA 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". In one aspect,
host cells include prokaryotic and eukaryotic cells selected from
any of the Kingdoms of life. Eukaryotic cells include protist,
fungal, plant and animal cells. In another embodiment, host cells
include, but are not limited, to the prokaryotic cell line
Escherichia coli; mammalian cell lines CHO, HEK 293 and COS; the
insect cell line Sf9; and the fungal cell Saccharomyces
cerevisiae.
[0119] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation and lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual, 2d ed. (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. 1989).
[0120] The term "transgenic organism" refers to an organism having
cells that contain a transgene, wherein the transgene introduced
into the organism (or an ancestor of the organism) expresses a
polypeptide not naturally expressed in the organism. A "transgene"
is a DNA construct that is stably and operably integrated into the
genome of a cell from which a transgenic organism develops,
directing the expression of an encoded gene product in one or more
cell types or tissues of the transgenic organism.
[0121] The terms "regulate" and "modulate" are used interchangeably
and refer to a change or an alteration in the activity of a
molecule of interest (e.g., the biological activity of
hIL-1.beta.). Modulation may be an increase or a decrease in the
magnitude of a certain activity or function of the molecule of
interest. Exemplary activities and functions of a molecule include,
but are not limited to, binding characteristics, enzymatic
activity, cell receptor activation, and signal transduction.
[0122] Correspondingly, the term "modulator," is a compound capable
of changing or altering an activity or function of a molecule of
interest (e.g., the biological activity of hIL-1.alpha.). For
example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Exemplary inhibitors include, but are
not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies are
described, e.g., in PCT Publication No. WO01/83525.
[0123] The term "agonist" refers to a modulator that, when
contacted with a molecule of interest, causes an increase in the
magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in
the absence of the agonist. Particular agonists of interest may
include, but are not limited to, IL-1.beta. polypeptides or
polypeptides, nucleic acids, carbohydrates, or any other molecules
that bind to IL-1.beta..
[0124] The term "antagonist" or "inhibitor" refer to a modulator
that, when contacted with a molecule of interest, causes a decrease
in the magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in
the absence of the antagonist. Antagonists include those that block
or modulate the biological or immunological activity of IL-1.beta..
Antagonists and inhibitors of IL-1.beta. may include, but are not
limited to, proteins, nucleic acids, carbohydrates, or any other
molecules that bind to IL-1.beta..
[0125] The term "effective amount" refers to the amount of a
therapy that is sufficient to reduce or ameliorate the severity
and/or duration of a disorder or one or more symptoms thereof,
prevent the advancement of a disorder, cause regression of a
disorder, prevent the recurrence, development, onset or progression
of one or more symptoms associated with a disorder, detect a
disorder, or enhance or improve the prophylactic or therapeutic
effect(s) of another therapy (e.g., prophylactic or therapeutic
agent).
[0126] The term "sample" is used in its broadest sense. A
"biological sample" includes, but is not limited to, any quantity
of a substance from a living thing or formerly living thing. Such
living things include, but are not limited to, humans, mice, rats,
monkeys, dogs, rabbits and other animals. Such substances include,
but are not limited to, blood, serum, urine, synovial fluid, cells,
organs, tissues, bone marrow, lymph nodes and spleen.
I. Antibodies that Bind Human IL-1.alpha.
[0127] One aspect of the present invention provides isolated
monoclonal antibodies, or antigen-binding portions thereof, that
bind to IL-1.beta. with high affinity, have a slow off rate and
have a high neutralizing capacity. A second aspect of the invention
provides chimeric antibodies that bind IL-1.beta.. A third aspect
of the invention provides CDR grafted antibodies, or
antigen-binding portions thereof, that bind IL-1.beta.. A fourth
aspect of the invention provides humanized antibodies, or
antigen-binding portions thereof, that bind IL-1.beta.. In an
embodiment, the antibodies, or portions thereof, are isolated
antibodies. In an embodiment, the antibodies of the invention are
neutralizing human anti-IL-1.beta. antibodies.
A. Methods of Making Anti-IL-1.beta. Antibodies
[0128] Antibodies of the present invention may be made by any of a
number of techniques known in the art.
1. Anti-IL-1.beta. Monoclonal Antibodies Using Hybridoma
Technology
[0129] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow and Lane, Antibodies: A Laboratory Manual,
2d ed., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
1988); Hammerling et al., eds., "Monoclonal Antibodies and T-Cell
Hybridomas," In Research Monographs in Immunology, vol. 3 (J. L.
Turk, General Editor) (Elsevier, N.Y., 1981) pp. 563-587. The term
"monoclonal antibody" is not limited to antibodies produced through
hybridoma technology. The term "monoclonal antibody" refers to an
antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced.
[0130] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In one embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein the hybridoma is generated by
fusing splenocytes isolated from a mouse immunized with an antigen
of the invention with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind a polypeptide of the invention.
Briefly, mice can be immunized with an IL-1.beta. antigen. In a
particular embodiment, the IL-1.alpha. antigen is administered with
an adjuvant to stimulate the immune response. Such adjuvants
include complete or incomplete Freund's adjuvant, RIBI (muramyl
dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants
may protect the polypeptide from rapid dispersal by sequestering it
in a local deposit, or they may contain substances that stimulate
the host to secrete factors that are chemotactic for macrophages
and other components of the immune system. In an embodiment, if a
polypeptide is being administered, the immunization schedule will
involve two or more administrations of the polypeptide, spread out
over several weeks.
[0131] After immunization of an animal with an IL-1.beta. antigen,
antibodies and/or antibody-producing cells may be obtained from the
animal. An anti-IL-1.beta. antibody-containing serum is obtained
from the animal by bleeding or sacrificing the animal. The serum
may be used as it is obtained from the animal, an immunoglobulin
fraction may be obtained from the serum, or the anti-IL-1.beta.
antibodies may be purified from the serum. Serum or immunoglobulins
obtained in this manner are polyclonal, thus having a heterogeneous
array of properties.
[0132] Once an immune response is detected, e.g., antibodies
specific for the antigen IL-1.beta. are detected in the mouse
serum, the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by well-known techniques to any suitable
myeloma cells, for example cells from cell line SP20 available from
the ATCC. Hybridomas are selected and cloned by limited dilution.
The hybridoma clones are then assayed by methods known in the art
for cells that secrete antibodies capable of binding IL-1.beta..
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0133] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
(See, e.g., Harlow and Lane, supra). In a particular embodiment,
the myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using IL-1.beta., or a portion thereof,
or a cell expressing IL-1.beta.. In a particular embodiment, the
initial screening is performed using an enzyme-linked immunoassay
(ELISA) or a radioimmunoassay (RIA. An example of ELISA screening
is provided in PCT Publication No. WO 00/37504.
[0134] Anti-IL-1.beta. antibody-producing hybridomas are selected,
cloned and further screened for desirable characteristics,
including robust hybridoma growth, high antibody production and
desirable antibody characteristics, as discussed further below.
Hybridomas may be cultured and expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0135] In an embodiment, the hybridomas are mouse hybridomas. In
another embodiment, the hybridomas are produced in a non-human,
non-mouse species such as rats, sheep, pigs, goats, cattle or
horses. In yet another embodiment, the hybridomas are human
hybridomas, in which a human non-secretory myeloma is fused with a
human cell expressing an anti-IL-1.beta. antibody.
[0136] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
2. Anti-IL-1.beta. Monoclonal Antibodies Using SLAM
[0137] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication
No. WO 92/02551 and Babcook et al. (1996) Proc. Natl. Acad. Sci.
USA 93:7843-7848. In this method, single cells secreting antibodies
of interest, e.g., lymphocytes derived from an immunized animal,
are screened using an antigen-specific hemolytic plaque assay,
wherein the antigen IL-1.beta., or a fragment thereof, is coupled
to sheep red blood cells using a linker, such as biotin, and used
to identify single cells that secrete antibodies with specificity
for IL-1.beta.. Following identification of antibody-secreting
cells of interest, heavy- and light-chain variable region cDNAs are
rescued from the cells by reverse transcriptase-PCR and these
variable regions can then be expressed, in the context of
appropriate immunoglobulin constant regions (e.g., human constant
regions), in mammalian host cells, such as COS or CHO cells. The
host cells transfected with the amplified immunoglobulin sequences,
derived from in vivo selected lymphocytes, can then undergo further
analysis and selection in vitro, for example by panning the
transfected cells to isolate cells expressing antibodies to
IL-1.beta.. The amplified immunoglobulin sequences further can be
manipulated in vitro, such as by in vitro affinity maturation
methods such as those described in PCT Publication Nos. WO 97/29131
and WO 00/56772.
3. Anti-IL-1.beta. Monoclonal Antibodies Using Transgenic
Animals
[0138] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with an IL-1.beta. antigen.
In an embodiment, the non-human animal is a XENOMOUSE.RTM.
transgenic mouse, an engineered mouse strain that comprises large
fragments of the human immunoglobulin loci and is deficient in
mouse antibody production. See, e.g., Green et al. (1994) Nature
Genet. 7:13 21 and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615;
5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364. See also
PCT Publication Nos. WO 91/10741; WO 94/02602; WO 96/34096; WO
96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO 99/45031; WO
99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE.RTM.
transgenic mouse produces an adult-like human repertoire of fully
human antibodies, and generates antigen-specific human monoclonal
antibodies. The XENOMOUSE.RTM. transgenic mouse contains
approximately 80% of the human antibody repertoire through
introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and x light chain loci.
See, Mendez et al. (1997) Nature Genet. 15:146-156 and Green and
Jakobovits (1998) J. Exp. Med. 188:483-495.
4. Anti-IL-1.beta. Monoclonal Antibodies Using Recombinant Antibody
Libraries
[0139] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, U.S. Pat.
No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO
92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; WO
97/29131; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al. (1990) Nature 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) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nucl. Acids Res. 19:4133-4137; and Barbas
et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982, and U.S.
Patent Publication No. 2003/0186374.
[0140] The recombinant antibody library may be from a subject
immunized with IL-1.beta., or a portion of IL-1.beta..
Alternatively, the recombinant antibody library may be from a naive
subject, i.e., one who has not been immunized with IL-1.beta., such
as a human antibody library from a human subject who has not been
immunized with human IL-1.beta.. Antibodies of the invention are
selected by screening the recombinant antibody library with the
peptide comprising human IL-1.beta. to thereby select those
antibodies that recognize IL-1.beta.. Methods for conducting such
screening and selection are well known in the art, such as
described in the references in the preceding paragraph. To select
antibodies of the invention having particular binding affinities
for hIL-1.beta., such as those that dissociate from human
IL-1.beta. with a particular k.sub.off rate constant, the art-known
method of surface plasmon resonance can be used to select
antibodies having the desired k.sub.off rate constant. To select
antibodies of the invention having a particular neutralizing
activity for hIL-1.beta., such as those with a particular
IC.sub.50, standard methods known in the art for assessing the
inhibition of hIL-1.beta. activity may be used.
[0141] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds human
IL-1.beta.. In a particular embodiment, the antibody is a
neutralizing antibody. In various embodiments, the antibody is a
recombinant antibody or a monoclonal antibody.
[0142] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkmann et al. (1995) J. Immunol. Methods 182:41-50; Ames et al.
(1995) J. Immunol. Methods 184:177-186; Kettleborough et al. (1994)
Eur. J. Immunol. 24:952-958; Persic et al. (1997) Gene 187 9-18;
Burton et al. (1994) Adv. Immunol. 57:191-280; PCT Application No.
PCT/GB91/01134; PCT Publication Nos. WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108.
[0143] After phage selection, the antibody coding regions from the
phage can be isolated and used to generate whole antibodies
including human antibodies or any other desired antigen binding
fragment, and expressed in any desired host, including mammalian
cells, insect cells, plant cells, yeast, and bacteria, e.g., as
described in detail herein. For example, techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
PCT Publication No. WO 92/22324; Mullinax et al. (1992)
BioTechniques 12(6):864-869; Sawai et al. (1995) Am. J. Reprod.
Immunol. 34:26-34; and Better et al. (1988) Science 240:1041-1043.
Examples of techniques that can be used to produce single-chain Fvs
and antibodies include those described in U.S. Pat. Nos. 4,946,778
and 5,258,498; Huston et al. (1991) Methods Enzymol. 203:46-88; Shu
et al. (1993) Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra
et al. (1988) Science 240:1038-1041.
[0144] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 and in Roberts and
Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this
system, a covalent fusion is created between an mRNA and the
peptide or protein that it encodes by in vitro translation of
synthetic mRNAs that carry puromycin, a peptidyl acceptor
antibiotic, at their 3' end. Thus, a specific mRNA can be enriched
from a complex mixture of mRNAs (e.g., a combinatorial library)
based on the properties of the encoded peptide or protein, e.g.,
antibody, or portion thereof, such as binding of the antibody, or
portion thereof, to the dual specificity antigen. Nucleic acid
sequences encoding antibodies, or portions thereof, recovered from
screening of such libraries can be expressed by recombinant means
as described above (e.g., in mammalian host cells) and, moreover,
can be subjected to further affinity maturation by either
additional rounds of screening of mRNA-peptide fusions in which
mutations have been introduced into the originally selected
sequence(s), or by other methods for affinity maturation in vitro
of recombinant antibodies, as described above.
[0145] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed U.S. Pat. No.
6,699,658.
B. Production of Recombinant IL-1.beta. Antibodies
[0146] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) 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 possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is preferable, and most preferable in mammalian
host cells, 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.
[0147] Exemplary 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 Kaufman and
Sharp (1982) J. 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 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.
[0148] Host cells can also be used to produce functional antibody
fragments, 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 functional fragments of
either the light chain and/or the heavy chain 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 the
antigens of interest. 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 human IL-1.beta. by crosslinking an antibody of the
invention to a second antibody by standard chemical crosslinking
methods.
[0149] In a particular system for recombinant expression of an
antibody of the invention, or antigen-binding portion thereof, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured 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.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
1. Anti IL-1.beta. Antibodies
[0150] Table 5 provides the amino acid sequences of VH and VL
regions of the mouse anti-hIL-1.beta. monoclonal antibody (mAb) of
the invention.
TABLE-US-00005 TABLE 5 Amino Acid Sequences of VH and VL Regions of
Mouse Anti-Human IL-1.beta. SEQ ID Amino Acid Sequence No. Protein
region 123456789012345678901234567890 26 1B12.4H4 VH
QVHLKESGPGLVAPSQSLSITCTVSGFSLT DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN
SPLKSRLSIRKDNSKSQVFLKMNSLQTDDT AVYYCAKQRTLWGYDLYGMDYWGQGTSVTV SS 27
1B12.4H4 VL ETTVTQSPASLSMAIGEKVTIRCITSTDID
VDMNWYQQKPGEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLSEDVADYYCLQ
SDNLPLTFGAGTKLELK
2. Anti IL-1.beta. Chimeric Antibodies
[0151] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art and
are discussed in the Examples. See, e.g., Morrison (1985) Science
229:1202-1207; Oi et al. (1986) BioTechniques 4:214-221; Gillies et
al. (1989) J. Immunol. Methods 125:191-202; and U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397. In addition, techniques
developed for the production of "chimeric antibodies" by splicing
genes from a mouse antibody molecule of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological activity can be used (Morrison et al. (1984)
Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger et al. (1984)
Nature 312:604-608; and Takeda et al. (1985) Nature
314:452-454).
3. Anti-IL-1.beta. CDR-Grafted Antibodies
[0152] CDR-grafted antibodies of the invention comprise heavy and
light chain variable region sequences from a human antibody wherein
one or more of the CDR regions of VH and/or VL are replaced with
CDR sequences of the murine antibodies of the invention. A
framework sequence from any human antibody may serve as the
template for CDR grafting. However, straight chain replacement onto
such a framework often leads to some loss of binding affinity to
the antigen. The more homologous a human antibody is to the
original murine antibody, the less likely the possibility that
combining the murine CDRs with the human framework will introduce
distortions in the CDRs that could reduce affinity. Therefore, in
an embodiment, the human variable framework that is chosen to
replace the murine variable framework apart from the CDRs have at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, about 100%, sequence identity with the murine antibody
variable region framework. Methods for producing CDR-grafted
antibodies are known in the art and described in detail along with
humanization of such CDR-grafted antibodies in the Examples (see
also, EP Patent No. EP 0 239 400; PCT Publication No. WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089); veneering or
resurfacing (EP Patent Nos. EP 0 592 106 and EP 0 519 596; Padlan
(1991) Mol. Immunol. 28(4/5):489-498; Studnicka et al. (1994)
Protein Eng. 7(6):805-814; Roguska et al. (1994) Proc. Natl. Acad.
Sci. USA 91:969-973), and chain shuffling (U.S. Pat. No.
5,565,352).
4. Anti IL-1.beta. Humanized Antibodies
[0153] Humanized antibodies are antibody molecules that have one or
more complementarity determining regions (CDRs) from a non-human
species and framework regions from a human immunoglobulin molecule.
Known human Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez-/query.fcgi;
www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uni-heidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH-05/kuby05.htm;
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.-html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html-;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/lin-ks.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;
baserv.uci.kun.nl/.about.jraats/links1.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;
www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.unizh.ch/.about.honegger/AHOsem-inar/Slide01.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.about.finolina/Web-pages/Pept/spottech.html;
www.jerini.de/frroducts.htm; www.patents.ibm.com/ibm.html.Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983). Such imported sequences can be used to reduce
immunogenicity or reduce, enhance or modify binding, affinity,
on-rate, off-rate, avidity, specificity, half-life, or any other
suitable characteristic, as known in the art.
[0154] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, for example, improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., U.S. Pat. No.
5,585,089 and Riechmann et al. (1988) Nature 332:323-327).
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available that illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as but not limited to those described in Jones et al.
(1986) Nature 321:522-525; Verhoeyen et al. (1988) Science
239:1534-1536), Sims et al. (1993) J. Immunol. 151: 2296-2308;
Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Carter et al.
(1992) Proc. Natl. Acad. Sci. USA 89:4285-4289; Presta et al.
(1993) J. Immunol. 151:2623-2632; Padlan (1991) Mol. Immunol.
28(4/5):489-498; Studnicka et al. (1994) Protein Eng. 7(6):805-814;
Roguska. et al. (1994) Proc. Natl. Acad. Sci. USA 91:969-973; PCT
Publication Nos. WO 91/09967, WO 99/06834 (PCT/US98/16280), WO
97/20032 (PCT/US96/18978), WO 92/11272 (PCT/US91/09630), WO
92/03461 (PCT/US91/05939), WO 94/18219 (PCT/US94/01234), WO
92/01047 (PCT/GB91/01134), WO 93/06213 (PCT/GB92/01755), WO
90/14443, WO 90/14424, and WO 90/14430; European Publication Nos.
EP 0592106, EP 0519596, and EP 0239400; U.S. Pat. Nos. 5,565,332;
5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192;
5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762;
5,530,101; 5,585,089; 5,225,539; and 4,816,567.
C. Production of Antibodies and Antibody-Producing Cell Lines
[0155] In an embodiment, anti-IL-1.beta. antibodies of the present
invention, exhibit a high capacity to reduce or to neutralize
IL-1.beta. activity, e.g., as assessed by any one of several in
vitro and in vivo assays known in the art. In an embodiment,
anti-IL-1.beta. antibodies of the present invention, also exhibit a
high capacity to reduce or to neutralize IL-1.beta. activity
[0156] In particular embodiments, the isolated antibody, or
antigen-binding portion thereof, binds human IL-1.beta., wherein
the antibody, or antigen-binding portion thereof, dissociates from
human IL-1.beta. with a k.sub.off rate constant of about 0.1
s.sup.-1 or less, as determined by surface plasmon resonance, or
which inhibits human IL-1.beta. activity with an IC.sub.50 of about
1.times.10.sup.-6M or less. Alternatively, the antibody, or an
antigen-binding portion thereof, may dissociate from human
IL-1.beta. with a k.sub.off rate constant of about
1.times.10.sup.-2 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-1.beta. activity with an
IC.sub.50 of about 1.times.10.sup.-7M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-1.beta. with a k.sub.off rate constant of about
1.times.10.sup.-3 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-1.beta. with an
IC.sub.50 of about 1.times.10.sup.-8M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-1.beta. with a k.sub.off rate constant of about
1.times.10.sup.-4 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit IL-1.beta. activity with an
IC.sub.50 of about 1.times.10.sup.-9M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-1.beta. with a k.sub.off rate constant of about
1.times.10.sup.-5 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit IL-1.beta. activity with an
IC.sub.50 of about 1.times.10.sup.-10M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-1.beta. with a k.sub.off rate constant of about
1.times.10.sup.-5 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-1.beta. activity with an
IC.sub.50 of about 1.times.10.sup.-11M or less.
[0157] 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. In an embodiment, 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. In an embodiment,
the antibody comprises a kappa light chain constant region.
Alternatively, the antigen binding portion can be, for example, a
Fab fragment or a single chain Fv fragment.
[0158] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (U.S. Pat.
Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody
mediates several important effector functions, e.g., cytokine
induction, antibody dependent cell-mediated cytotoxicity (ADCC),
phagocytosis, complement dependent cytotoxicity (CDC) and
half-life/clearance rate of antibody and antigen-antibody
complexes. In some cases these effector functions are desirable for
a therapeutic antibody but in other cases might be unnecessary or
even deleterious, depending on the therapeutic objectives. Certain
human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and
CDC via binding to Fc.gamma.Rs and complement C1q, respectively.
Neonatal Fc receptors (FcRn) are the critical components
determining the circulating half-life of antibodies. In still
another embodiment at least one amino acid residue is replaced in
the constant region of the antibody, for example the Fc region of
the antibody, such that effector functions of the antibody are
altered.
[0159] One embodiment provides a labeled binding protein wherein an
antibody, or antigen binding portion thereof, of the invention is
derivatized or linked to another functional molecule (e.g., another
peptide or protein). For example, a labeled binding protein of the
invention can be derived by functionally linking an antibody, or
antigen binding portion thereof, of the invention (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 association of the antibody, or antigen
binding portion thereof, with another molecule (such as a
streptavidin core region or a polyhistidine tag).
[0160] Useful detectable agents with which an antibody, or antigen
binding portion thereof, 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.
[0161] Another embodiment of the invention provides a crystallized
binding protein. In an embodiment, the invention relates to
crystals of whole anti-IL-1.beta. antibodies and fragments thereof
as disclosed herein, and formulations and compositions comprising
such crystals. In one embodiment the crystallized binding protein
has a greater half-life in vivo than the soluble counterpart of the
binding protein. In another embodiment the binding protein retains
biological activity after crystallization.
[0162] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in PCT
Publication No. WO 02/72636.
[0163] Another embodiment of the invention provides a glycosylated
binding protein wherein an antibody, or antigen-binding portion
thereof, comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Protein glycosylation depends on the amino acid
sequence of the protein of interest, as well as the host cell in
which the protein is expressed. Different organisms may produce
different glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (e.g., nucleotide
sugars) available. Due to such factors, protein glycosylation
pattern, and composition of glycosyl residues, may differ depending
on the host system in which the particular protein is expressed.
Glycosyl residues useful in the invention may include, but are not
limited to, glucose, galactose, mannose, fucose,
n-acetylglucosamine and sialic acid. In an embodiment, the
glycosylated binding protein comprises glycosyl residues such that
the glycosylation pattern is human.
[0164] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0165] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (U.S. Pat. Nos. 7,449,308 and
7,029,872).
[0166] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. In an embodiment, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
D. Uses of Anti-IL-1.beta. Antibodies
[0167] Given their ability to bind to human IL-1.beta., the binding
proteins, e.g., anti-IL-1.beta. antibodies and antigen-binding
portions thereof, according to the invention can be used to detect
IL-1.beta. (e.g., in a biological sample, such as whole blood,
serum, plasma, urine, saliva, tissue sample) using any of the vast
array of antibody-based immunodetection systems available in the
art. Such immunodetection systems include, but are not limited to,
immunoprecipitation, immunblotting (Western blot), enzyme-linked
immunsorbent assay (ELISA), radioimmunoassay (RIA), tissue
immunohistochemistry, surface plasmon resonance (SPR), sandwich
immunoassay, antibody-based affinity methods (e.g., affinity beads,
affinity columns), immunocompetition assay, immunochip assay
(binding protein attached to a silicon chip), and fluorescence
activated cell sorting (FACS). For some immunodetection systems, an
IL-1.beta. binding protein (or binding portion thereof) of the
invention (or portion thereof) is attached to a solid substrate
using methods available in the art for attaching antibody molecules
to the same solid substrate so that the attached binding protein
retains its ability to bind human IL-1.beta. during use in the
particular immunodetection system. Such solid substrates include,
but are not limited to, a cellulose-based filter paper (e.g.,
cellulose, nitrocellulose, cellulose acetate), a nylon filter, a
plastic surface (e.g., microtiter plate, antibody dip stick), a
glass substrate (e.g., filters, beads, slides, glass wool), a
polymeric particle (e.g., agarose, polyacrylamide), and a silicon
chip. For example, an immunodetection system may be used in a
method for detecting the presence of IL-1.beta. in a sample in
vitro (e.g., a biological sample, such as whole blood, serum,
plasma, tissue, urine, saliva, tissue biopsy). Such a method can be
used to diagnose a disease or disorder, e.g., an immune
cell-associated disorder. The method includes: (i) contacting a
test sample or a control sample with an IL-1.beta. binding protein,
or IL-1.beta. binding portion thereof, as described herein; and
(ii) detecting formation of a complex between the anti-IL-1.beta.
binding protein (or binding portion thereof) and IL-1.beta. in the
test sample or in the control sample, wherein a statistically
significant change in the formation of the complex in the test
sample relative to the control sample (or relative to formation of
the complex in another test sample taken at an earlier time point)
is indicative of the presence of IL-1.beta. in the sample.
[0168] As another example, a method may be employed for detecting
the presence of human IL-1.beta. in vivo (e.g., in vivo imaging in
a subject). The method can be used to diagnose a disease or
disorder, e.g., an IL-1.beta.-associated disorder. The method
includes: (i) administering an IL-1.beta. binding protein, or
IL-1.beta. binding portion thereof, as described herein to a test
subject or a control subject under conditions that allow binding of
the binding protein, or IL-1.beta. binding portion thereof, to
IL-1.beta.; and (ii) detecting formation of a complex between the
binding protein, or binding portion thereof, and IL-1.beta.,
wherein a statistically significant change in the formation of the
complex in the test subject relative to the control subject, or
relative to formation of the complex in the test subject at an
earlier time point, is indicative of the presence of IL-1.beta..
Methods for detecting IL-1.beta. in a sample (e.g., a biological
sample) according to the invention comprise contacting a sample
with an IL-1.beta. binding protein (or IL-1.beta. binding portion
thereof) described herein and detecting either the binding protein
(or binding portion thereof) bound to IL-1.beta. or unbound binding
protein (or unbound binding portion thereof) to thereby detect
IL-1.beta. in the sample. The binding protein (or portion thereof)
is directly or indirectly labeled with a detectable substance to
facilitate detection of the bound or unbound binding protein (or
portion thereof). Such detectable substances are known in the art
and, by way of non-limiting example, 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. Examples of suitable radioactive materials include the
radioisotopes 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu,
166Ho, and 153Sm. Alternatively to labeling the binding protein,
human IL-1.beta. can be assayed in a sample (e.g., a biological
fluid) by a competition immunoassay utilizing recombinant human
(rh) IL-1.beta. standards labeled with a detectable substance and
an unlabeled IL-1.beta. binding protein (or IL-1.beta. binding
portion thereof). In this assay, the sample, the labeled rh
IL-1.beta. standards, and the IL-1.beta. binding protein are
combined and the amount of labeled rh IL-1.beta. standard bound to
the unlabeled binding protein is determined. The amount of human
IL-1.beta. in the sample is inversely proportional to the amount of
labeled rh IL-1.beta. standard bound to the IL-1.beta. binding
protein. Similarly, human IL-1.beta. can also be assayed in a
sample by a competition immunoassay utilizing rh IL-1.beta.
standards labeled with a detectable substance and an unlabeled
IL-1.beta. binding protein described herein.
[0169] In an embodiment, the IL-1.beta. binding proteins, and
IL-1.beta. binding portions thereof, according to the invention are
capable of neutralizing IL-1.beta. activity both in vitro and in
vivo. Accordingly, such binding proteins, and IL-1.beta. binding
portions thereof, of the invention can be used to inhibit
IL-1.beta. activity, e.g., in a cell culture containing IL-1.beta.,
in human subjects, or in other mammalian subjects having IL-1.beta.
with which a binding protein of the invention cross-reacts. In one
embodiment, the invention provides a method for inhibiting
IL-1.beta. activity comprising contacting IL-1.beta. with a binding
protein, or binding portion thereof, of the invention such that
IL-1.beta. activity is inhibited. For example, in a cell culture
containing or suspected of containing IL-1.beta., a binding protein
(or binding portion thereof) of the invention can be added to the
culture medium to inhibit IL-1.beta. activity in the culture.
[0170] In another embodiment, the invention provides a method for
reducing IL-1.beta. activity in a subject, advantageously from a
subject suffering from a disease or disorder in which IL-1.beta.
activity is detrimental. The invention provides methods for
reducing IL-1.beta. activity in a subject suffering from such a
disease or disorder, which method comprises administering to the
subject a binding protein or antigen binding portion thereof, of
the invention such that IL-1.beta. activity in the subject is
reduced. In an embodiment, the IL-1.beta. is human IL-1.beta., and
the subject is a human subject. Alternatively, the subject can be a
mammal expressing an IL-1.beta. to which a binding protein of the
invention is capable of binding. Still further the subject can be a
mammal into which IL-1.beta. has been introduced (e.g., by
administration of IL-1.beta. or by expression of an IL-1.beta.
transgene). A binding protein of the invention can be administered
to a human subject for therapeutic purposes. Moreover, a binding
protein of the invention can be administered to a non-human mammal
expressing an IL-1.beta. with which the binding protein is capable
of binding 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).
[0171] The term "a disorder in which IL-1.beta. activity is
detrimental" includes diseases and other disorders in which the
presence of IL-1.beta. 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
IL-1.beta. activity is detrimental is a disorder in which reduction
of IL-1.beta. 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 IL-1.beta. in a
biological fluid of a subject suffering from the disorder (e.g., an
increase in the concentration of IL-1.beta. in serum, plasma,
synovial fluid, etc. of the subject), which can be detected, for
example, using an anti-IL-1.beta. binding protein as described
above. Non-limiting examples of disorders that can be treated with
the binding proteins of the invention include those disorders
discussed in the section below pertaining to pharmaceutical
compositions of the binding proteins of the invention.
E. Pharmaceutical Compositions
[0172] The invention also provides pharmaceutical compositions
comprising a binding protein (e.g., an antibody, or antigen-binding
portion thereof) of the invention and a pharmaceutically acceptable
carrier. The pharmaceutical compositions comprising binding
proteins of the invention are for use in, but not limited to,
diagnosing, detecting, or monitoring a disorder, in preventing,
treating, inhibiting, managing, or ameliorating of a disorder or
one or more symptoms thereof, and/or in research. In a specific
embodiment, a composition comprises one or more binding proteins of
the invention. In another embodiment, the pharmaceutical
composition comprises one or more binding proteins of the invention
and one or more prophylactic or therapeutic agents other than the
one or more binding proteins of the invention for treating a
disorder in which IL-1.beta. activity is detrimental. In
particular, the prophylactic or therapeutic agents are known to be
useful for, or have been, or are currently being used in the
prevention, treatment, management, or amelioration of a disorder,
or one or more symptoms thereof. In accordance with these
embodiments, the composition may further comprise of a carrier,
diluent or excipient.
[0173] The binding proteins of the invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises a
binding protein (e.g., an antibody or antigen binding portion
thereof) of the invention and a pharmaceutically acceptable
carrier. The term "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. It may 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 antigen binding portion
thereof.
[0174] Various delivery systems are known and can be used to
administer one or more binding proteins of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu
and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods
of administering a prophylactic or therapeutic agent of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration,
intratumoral administration, and mucosal administration (e.g.,
intranasal and oral routes). In addition, pulmonary administration
can be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913;
5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO
97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In one
embodiment, a binding protein of the invention, combination
therapy, or a composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.). The prophylactic or therapeutic agents may
be administered by any convenient route, for example by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and
may be administered together with other biologically active agents.
Administration can be systemic or local.
[0175] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment, for example, by local infusion, by
injection, or by means of an implant. An implant may be porous or
non-porous material, including membranes and matrices, such as
sialastic membranes, polymers, fibrous matrices (e.g.,
TISSUEL.RTM.), or collagen matrices. In one embodiment, an
effective amount of one or more antibodies of the invention
antagonists is administered locally to the affected area to a
subject to prevent, treat, manage, and/or ameliorate a disorder or
a symptom thereof. In another embodiment, an effective amount of
one or more antibodies of the invention is administered locally to
the affected area in combination with an effective amount of one or
more therapies (e.g., one or more prophylactic or therapeutic
agents) other than a binding protein of the invention of a subject
to prevent, treat, manage, and/or ameliorate a disorder or one or
more symptoms thereof.
[0176] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release (see Langer (1990) Science 249:1527-1533;
Sefton (1987) CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et
al. (1980) Surgery 88:507-516; Saudek et al. (1989) N. Engl. J.
Med. 321:574-579). In another embodiment, polymeric materials can
be used to achieve controlled or sustained release of the therapies
of the invention (see, e.g., Medical Applications of Controlled
Release, (Langer and Wise, eds.) (CRC Press, Inc., Boca Raton,
1984); Controlled Drug Bioavailability, Drug Product Design and
Performance, (Smolen and Ball, eds.) (Wiley, New York, 1984);
Langer and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem.
Phys. C23:61-126; see also Levy et al. (1985) Science 228:190-192;
During et al. (1989) Ann. Neurol. 25:351-356; Howard et al. (1989)
J. Neurosurg. 71:105-112); U.S. Pat. Nos. 5,679,377; 5,916,597;
5,912,015; 5,989,463; and 5,128,326; and PCT Publication Nos. WO
99/15154 and WO 99/20253. Examples of polymers used in sustained
release formulations include, but are not limited to, poly(-hydroxy
ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In an embodiment, the polymer used in a sustained
release formulation is inert, free of leachable impurities, stable
on storage, sterile, and biodegradable. In yet another embodiment,
a controlled or sustained release system can be placed in proximity
of the prophylactic or therapeutic target, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, J. M., Chapter
6, In Medical Applications of Controlled Release, Vol. II,
Applications and Evaluation, (Langer and Wise, eds.)(CRC Press,
Inc., Boca Raton, 1984), pp. 115-138).
[0177] Controlled release systems are discussed in the review by
Langer (1990) Science 249:1527-1533. Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; and PCT Publication
Nos. WO 91/05548 and WO 96/20698; and Ning et al. (1996) Radiother.
Oncol. 39:179-189; Song et al. (1996) PDA J. Pharm. Sci. Technol.
50:372-377; Cleek et al. (1997) Proceed Int'l. Symp. Control. Rel.
Bioact. Mater. 24:853-854; and Lam et al. (1997) Proceed. Int'l.
Symp. Control Rel. Bioact. Mater. 24:759-760.
[0178] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci.
USA 88:1864-1868). Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression by homologous recombination.
[0179] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0180] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed., (Mack
Publishing Co., Easton, Pa., 1995). For non-sprayable topical
dosage forms, viscous to semi-solid or solid forms comprising a
carrier or one or more excipients compatible with topical
application and having a dynamic viscosity preferably greater than
water are typically employed. Other suitable formulations include,
without limitation, suspensions, powders, liniments, salves, and
the like. In an embodiment, such formulations are sterilized or
mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, for example, in combination with a solid or
liquid inert carrier, is packaged in a mixture with a pressurized
volatile (e.g., a gaseous propellant, such as FREON.RTM.) or in a
squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples
of such additional ingredients are well-known in the art.
[0181] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0182] If the method of the invention comprises oral
administration, compositions can be formulated orally in the form
of tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0183] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0184] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0185] The methods of the invention may additionally comprise of
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0186] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0187] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0188] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. In an embodiment,
one or more of the prophylactic or therapeutic agents or
pharmaceutical compositions of the invention is supplied as a dry
sterile lyophilized powder in a hermetically sealed container at a
unit dosage of at least about 5 mg, at least about 10 mg, at least
about 15 mg, at least about 25 mg, at least about 35 mg, at least
about 45 mg, at least about 50 mg, at least about 75 mg, or at
least about 100 mg. The lyophilized prophylactic or therapeutic
agents or pharmaceutical compositions of the invention should be
stored at between about 2.degree. C. and about 8.degree. C. in its
original container and the prophylactic or therapeutic agents, or
pharmaceutical compositions of the invention should be administered
within 1 week, within 5 days, within 72 hours, within 48 hours,
within 24 hours, within 12 hours, within 6 hours, within 5 hours,
within 3 hours, or within 1 hour after being reconstituted. In an
alternative embodiment, one or more of the prophylactic or
therapeutic agents or pharmaceutical compositions of the invention
is supplied in liquid form in a hermetically sealed container
indicating the quantity and concentration of the agent. In an
embodiment, the liquid form of the administered composition is
supplied in a hermetically sealed container at least about 0.25
mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least
about 2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at
least about 10 mg/ml, at least about 15 mg/kg, at least about 25
mg/ml, at least about 50 mg/ml, at least about 75 mg/ml or at least
about 100 mg/ml. The liquid form should be stored at between about
2.degree. C. and about 8.degree. C. in its original container.
[0189] The binding proteins of the invention can be incorporated
into a pharmaceutical composition suitable for parenteral
administration. In one aspect, the binding proteins are prepared as
an injectable solution containing about 0.1 to about 250 mg/ml
antibody. The injectable solution can be composed of either a
liquid or lyophilized dosage form in a flint or amber vial, ampoule
or pre-filled syringe. The buffer can be L-histidine (about 1 to
about 50 mM), optimally about 5 to about 10 mM, at pH 5.0 to 7.0
(optimally about pH 6.0). Other suitable buffers include but are
not limited to, sodium succinate, sodium citrate, sodium phosphate
or potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of about 0 to about 300
mM (e.g., about 150 mM for a liquid dosage form). Cryoprotectants
can be included for a lyophilized dosage form, principally about 0
to about 10% sucrose (e.g., about 0.5 to about 1.0%). Other
suitable cryoprotectants include trehalose and lactose. Bulking
agents can be included for a lyophilized dosage form, principally
about 1 to about 10% mannitol (e.g., about 2 to about 4%).
Stabilizers can be used in both liquid and lyophilized dosage
forms, principally about 1 to about 50 mM L-Methionine (optimally
about 5 to about 10 mM). Other suitable bulking agents include
glycine, arginine, can be included as about 0 to about 0.05%
polysorbate-80 (optimally about 0.005 to about 0.01%). Additional
surfactants include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0190] 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 particular form depends
on the intended mode of administration and therapeutic application.
Typical 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 mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In an embodiment, the antibody is
administered by intravenous infusion or injection. In another
embodiment, the antibody is administered by intramuscular or
subcutaneous injection.
[0191] 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 (e.g., antibody or antigen
binding portion thereof) 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, lyophilized powders for the preparation of sterile
injectable solutions, the exemplary methods of preparation are
vacuum drying and spray-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.
[0192] The binding proteins of the present invention can be
administered by a variety of methods known in the art, although for
many therapeutic applications, an exemplary route/mode of
administration is subcutaneous injection, 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).
[0193] In certain embodiments, an antibody, or antigen binding
portion thereof, 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.
[0194] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, a bindin protein (e.g.,
an antibody) or antigen binding portion thereof, of the invention
is coformulated with and/or coadministered with one or more
additional therapeutic agents that are useful for treating
disorders in which IL-1.beta. activity is detrimental. For example,
an anti-hIL-1.beta. binding protein or antigen binding portion
thereof, 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). Furthermore, one or more binding proteins 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.
[0195] In certain embodiments, an IL-1.beta. binding protein, or
IL-1.beta.-binding portion thereof, as described herein is linked
to a half-life extending vehicle known in the art. Such vehicles
include, but are not limited to, the Fc domain, polyethylene
glycol, and dextran. Such vehicles are described, e.g., in U.S.
Pat. No. 6,660,843 and published PCT Publication No. WO
99/25044.
[0196] In a specific embodiment, nucleic acid molecules comprising
nucleotide sequences encoding one or more polypeptides of a binding
protein of the invention or another prophylactic or therapeutic
agent of the invention are administered to treat, prevent, manage,
or ameliorate a disorder or one or more symptoms thereof by way of
gene therapy. Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible nucleic
acid. In this embodiment of the invention, the nucleic acids
produce their encoded binding polypeptide(s) of a binding protein
or prophylactic or therapeutic agent of the invention that mediates
a prophylactic or therapeutic effect.
[0197] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al. (1993) Clin.
Pharm. 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev
(1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993)
Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev.
Biochem. 62:191-217; Robinson, C. (1993) Trends Biotechnol.
11(5):155. Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology (John Wiley &
Sons, New York, 1993); and Kriegler, Gene Transfer and Expression,
A Laboratory Manual, (Stockton Press, New York, 1990). Detailed
descriptions of various methods of gene therapy are disclosed in US
2005/0042664.
[0198] IL-1.beta. plays a critical role in the pathology associated
with a variety of diseases involving immune and inflammatory
elements. These diseases include, but are not limited to, Acquired
Immunodeficiency Disease Syndrome; Acquired Immunodeficiency
Related Diseases; acquired pernicious anaemia; acute coronary
syndromes; acute and chronic pain (different forms of pain); acute
idiopathic polyneuritis; acute immune disease associated with organ
transplantation; acute or chronic immune disease associated with
organ transplantation; acute inflammatory demyelinating
polyradiculoneuropathy; acute ischemia; acute liver disease; acute
rheumatic fever; acute transverse myelitis; Addison's disease;
adult (acute) respiratory distress syndrome; adult Still's disease;
alcoholic cirrhosis; alcohol-induced liver injury; allergic
diseases; allergy; alopecia; alopecia areata; Alzheimer's disease;
anaphylaxis; ankylosing spondylitis; ankylosing spondylitis
associated lung disease; anti-phospholipid antibody syndrome;
aplastic anemia; arteriosclerosis; arthropathy; asthma;
atheromatous disease/arteriosclerosis; atherosclerosis; atopic
allergy; atopic eczema; atopic dermatitis; atrophic autoimmune
hypothyroidism; autoimmune bullous disease; autoimmune dermatitis;
autoimmune diabetes; autoimmune disorder associated with
streptococcus infection; autoimmune enteropathy; autoimmune
haemolytic anaemia; autoimmune hepatitis; autoimmune hearing loss;
autoimmune lymphoproliferative syndrome (ALPS); autoimmune mediated
hypoglycaemia; autoimmune myocarditis; autoimmune neutropenia;
autoimmune premature ovarian failure; autoimmune thrombocytopenia
(AITP); autoimmune thyroid disease; autoimmune uveitis;
bronchiolitis obliterans; Behcet's disease; blepharitis;
bronchiectasis; bullous pemphigoid; cachexia; cardiovascular
disease; catastrophic antiphospholipid syndrome; celiac disease;
cervical spondylosis; chlamydia; choleosatatis; chronic active
hepatitis; chronic eosinophilic pneumonia; chronic fatigue
syndrome; chronic immune disease associated with organ
transplantation; chronic ischemia; chronic liver diseases; chronic
mucocutaneous candidiasis; cicatricial pemphigoid; clinically
isolated syndrome (CIS) with risk for multiple sclerosis; common
varied immunodeficiency (common variable hypogammaglobulinaemia);
connective tissue disease associated interstitial lung disease;
conjunctivitis; Coombs positive haemolytic anaemia; childhood onset
psychiatric disorder; chronic obstructive pulmonary disease (COPD);
Crohn's disease; cryptogenic autoimmune hepatitis; cryptogenic
fibrosing alveolitis; dacryocystitis; depression; dermatitis
scleroderma; dermatomyositis; dermatomyositis/polymyositis
associated lung disease; diabetic retinopathy; diabetes mellitus;
dilated cardiomyopathy; discoid lupus erythematosus; disk
herniation; disk prolapse; disseminated intravascular coagulation;
drug-induced hepatitis; drug-induced interstitial lung disease;
drug induced immune hemolytic anemia; endocarditis; endometriosis;
endophthalmitis; enteropathic synovitis; episcleritis; erythema
multiforme; erythema multiforme major; female infertility;
fibrosis; fibrotic lung disease; gestational pemphigoid; giant cell
arteritis (GCA); glomerulonephritides; goitrous autoimmune
hypothyroidism (Hashimoto's disease); Goodpasture's syndrome; gouty
arthritis; graft versus host disease (GVHD); Grave's disease; group
B streptococci (BGS) infection; Guillain-Barre syndrome (BGS);
haemosiderosis associated lung disease; hay fever; heart failure;
hemolytic anemia; Henoch-Schoenlein purpura; hepatitis B; hepatitis
C; Hughes syndrome; Huntington's chorea; hyperthyroidism;
hypoparathyroidism; idiopathic leucopaenia; idiopathic
thrombocytopaenia; idiopathic Parkinson's disease; idiopathic
interstitial pneumonia; idiosyncratic liver disease; IgE-mediated
allergy; immune hemolytic anemia; inclusion body myositis;
infectious diseases; infectious ocular inflammatory disease;
inflammatory bowel disease; inflammatory demyelinating disease;
inflammatory heart disease; inflammatory kidney disease; insulin
dependent diabetes mellitus; interstitial pneumonitis; IPF/UIP;
iritis; juvenile chronic arthritis; juvenile pernicious anaemia;
juvenile rheumatoid arthritis (JRA); Kawasaki's disease; keratitis;
keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meier
disease; Landry's paralysis; Langerhan's cell histiocytosis; linear
IgA disease; livedo reticularis; Lyme arthritis; lymphocytic
infiltrative lung disease; macular degeneration; male infertility
idiopathic or NOS; malignancies; microscopic vasculitis of the
kidneys; microscopic polyangiitis; mixed connective tissue disease
associated lung disease; Morbus Bechterev; motor neuron disorders;
mucous membrane pemphigoid; multiple sclerosis (all subtypes:
primary progressive, secondary progressive, relapsing remitting
etc.); multiple organ failure; myalgic encephalitis/royal free
disease; myasthenia gravis; myelodysplastic syndrome; myocardial
infarction; myocarditis; nephrotic syndrome; nerve root disorders;
neuropathy; non-alcoholic steatohepatitis; non A non B hepatitis;
optic neuritis; organ transplant rejection; osteoarthritis;
osteolysis; ovarian cancer; ovarian failure; pancreatitis;
parasitic diseases; Parkinson's disease; pauciarticular JRA;
pemphigoid; pemphigus foliaceus; pemphigus vulgaris; peripheral
artery occlusive disease (PAOD); peripheral vascular disease (PVD);
peripheral artery disease (PAD); phacogenic uveitis; phlebitis;
polyarteritis nodosa (or periarteritis nodosa); polychondritis;
polymyalgia rheumatica; poliosis; polyarticular JRA; polyendocrine
deficiency syndrome; polymyositis; polyglandular deficiency type I
and polyglandular deficiency type II; polymyalgia rheumatica (PMR);
postinfectious interstitial lung disease; post-inflammatory
interstitial lung disease; post-pump syndrome; premature ovarian
failure; primary biliary cirrhosis; primary myxoedema; primary
Parkinsonism; primary sclerosing cholangitis; primary sclerosing
hepatitis; primary vasculitis; prostate and rectal cancer and
hematopoietic malignancies (leukemia and lymphoma); prostatitis;
psoriasis; psoriasis type 1; psoriasis type 2; psoriatic arthritis;
psoriatic arthropathy; pulmonary hypertension secondary to
connective tissue disease; pulmonary manifestation of polyarteritis
nodosa; pure red cell aplasia; primary adrenal insufficiency;
radiation fibrosis; reactive arthritis; Reiter's disease; recurrent
neuromyelitis optica; renal disease NOS; restenosis; rheumatoid
arthritis; rheumatoid arthritis associated interstitial lung
disease; rheumatic heart disease; SAPHO (synovitis, acne,
pustulosis, hyperostosis, and osteitis); sarcoidosis;
schizophrenia; Schmidt's syndrome; scleroderma; secondary
amyloidosis; shock lung; scleritis; sciatica; secondary adrenal
insufficiency; sepsis syndrome; septic arthritis; septic shock;
seronegative arthropathy; silicone associated connective tissue
disease; Sjogren's disease associated lung disease; Sjorgren's
syndrome; Sneddon-Wilkinson dermatosis; sperm autoimmunity;
spondyloarthropathy; spondylitis ankylosans; Stevens-Johnson
syndrome (SJS); Still's disease; stroke; sympathetic ophthalmia;
systemic inflammatory response syndrome; systemic lupus
erythematosus; systemic lupus erythematosus associated lung
disease; systemic sclerosis; systemic sclerosis associated
interstitial lung disease; Takayasu's disease/arteritis; temporal
arteritis; Th2 Type and Th1 Type mediated diseases; thyroiditis;
toxic shock syndrome; toxoplasmic retinitis; toxic epidermal
necrolysis; transverse myelitis; TRAPS (Tumor-necrosis factor
receptor type 1 (TNFR)-Associated Periodic Syndrome); type B
insulin resistance with acanthosis nigricans; type 1 allergic
reaction; type-1 autoimmune hepatitis (classical autoimmune or
lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibody
hepatitis); type II diabetes; ulcerative colitic arthropathy;
ulcerative colitis; urticaria; usual interstitial pneumonia (UIP);
uveitis; vasculitic diffuse lung disease; vasculitis; vernal
conjunctivitis; viral retinitis; vitiligo; Vogt-Koyanagi-Harada
syndrome (VKH syndrome); Wegener's granulomatosis; wet macular
degeneration; wound healing; yersinia and salmonella associated
arthropathy.
[0199] In a particular embodiment, the IL-1.beta. binding proteins
and antigen-binding portions thereof of the invention are used to
treat rheumatoid arthritis, osteoarthritis, Crohn's disease,
multiple sclerosis, insulin dependent diabetes mellitus, and
psoriasis.
[0200] The IL-1.beta. binding proteins and antigen binding portions
thereof of the invention can also be used to treat humans suffering
from autoimmune diseases, in particular those associated with
inflammation, including ankylosing spondylitis, allergy, autoimmune
diabetes, and autoimmune uveitis.
[0201] An IL-1.beta. binding protein, or antigen binding portion
thereof, of the invention also can be administered with one or more
additional therapeutic agents useful in the treatment of autoimmune
and inflammatory diseases.
[0202] IL-1.beta. binding proteins of the invention, or antigen
binding portions thereof, can be used alone or in combination to
treat such diseases. It should be understood that the binding
proteins of the invention or antigen binding portions thereof can
be used alone or in combination with an additional agent, e.g., a
therapeutic agent, said additional agent being selected by the
skilled artisan for its intended purpose. For example, the
additional agent can be a therapeutic agent art-recognized as being
useful to treat the disease or condition being treated by the
antibody of the present invention. The additional agent also can be
an agent that imparts a beneficial attribute to the therapeutic
composition, e.g., an agent that affects the viscosity of the
composition.
[0203] The combinations of the invention include the IL-1.beta.
binding proteins, or antigen binding fragments thereof, described
herein and at least one additional agent listed below. The
combination can also include more than one additional agent, e.g.,
two or three additional agents if the combination is such that the
formed composition can perform its intended function.
[0204] Exemplary combinations include the IL-1.beta. binding
proteins, or antigen binding fragments thereof, described herein
and a non-steroidal anti-inflammatory drug(s) (NSAIDS), such as,
for example, ibuprofen. Other exemplary combinations comprise the
antibodies, or antigen binding fragments thereof, described herein
and corticosteroids including prednisolone. The side-effects of
steroid use can be reduced or eliminated by tapering the steroid
dose required when treating patients in combination with the
anti-IL-1.beta. binding proteins of this invention. Non-limiting
examples of therapeutic agents for rheumatoid arthritis with which
an antibody, or antibody portion, of the invention can be combined
include the following: cytokine suppressive anti-inflammatory
drug(s) (CSAIDs); antibodies to or antagonists of other human
cytokines or growth factors, for example, TNF, LT, IL-1.alpha.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18,
IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of
the invention, or antigen binding portions thereof, can be combined
with antibodies to cell surface molecules such as CD2, CD3, CD4,
CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2),
CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
[0205] Exemplary therapeutic agents for combining with the
IL-1.alpha. binding proteins, or antigen binding fragments thereof,
of the invention interfere at different points in the autoimmune
and subsequent inflammatory cascade, for example, TNF antagonists
like chimeric, humanized or human TNF antibodies, D2E7, (PCT
Publication No. WO 97/29131), CA2 (REMICADEa), CDP 571, and soluble
p55 or p75 TNF receptors, derivatives thereof, (p75TNFR1gG
(ENBRELa) or p55TNFR1gG (Lenercept), and also TNFa converting
enzyme (TACE) inhibitors, and other IL-1 inhibitors
(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.). Other
agents for combining with the antibodies and antigen binding
fragments thereof include Interleukin 11, agents that act parallel
to, dependent on, or in concert with IL-1a function such as, for
example, IL-18 antagonists (e.g., IL-18 binding proteins such as,
for example, antibodies or soluble IL-18 receptors, or antigen
binding fragments thereof. Additional agents for combining with the
antibodies and antigen binding fragments thereof include
non-depleting anti-CD4 inhibitors, antagonists of the
co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including
antibodies, soluble receptors, antagonistic ligands, or antigen
binding fragments thereof.
[0206] The binding proteins of the invention, or antigen binding
portions thereof, may also be combined with agents for treatment of
rheumatoid arthritis, for example, such as methotrexate, 6-MP,
azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, colchicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents that interfere with signaling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g., IRAK,
NIK, IKK, p38 and MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme (TACE) inhibitors,
T-cell signaling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g. soluble
p55 or p75 TNF receptors and the derivatives p75TNFRIgG (ENBREL.TM.
and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R),
antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGF.beta.), celecoxib, folic acid, hydroxychloroquine sulfate,
rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone
acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide,
propoxyphene napsylate/apap, folate, nabumetone, diclofenac,
piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl,
hydrocodone bitartrate/apap, diclofenac sodium/misoprostol,
fentanyl, anakinra, human recombinant, tramadol hcl, salsalate,
sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate
sodium, prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl,
sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram.
[0207] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which an IL-1.beta. binding protein (e.g., an
antibody), or antigen binding portion thereof, 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, antibodies to or antagonists of
other human cytokines or growth factors, for example, TNF, LT,
IL-1.beta., IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18,
EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or
antigen binding portions thereof, can be combined with antibodies
to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28,
CD30, CD40, CD45, CD69, CD90 and their ligands. The binding
proteins of the invention, or antigen binding portions thereof, may
also be combined with agents, such as methotrexate, cyclosporin,
FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for
example, ibuprofen, corticosteroids such as prednisolone,
phosphodiesterase inhibitors, adenosine agonists, antithrombotic
agents, complement inhibitors, adrenergic agents, agents which
interfere with signaling by proinflammatory cytokines such as
TNF.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme inhibitors, T-cell signaling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R)
and antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13,
and TGF.beta.).
[0208] Exemplary examples of therapeutic agents for Crohn's disease
in which an IL-1.beta. binding protein or an antigen binding
portion thereof, as described herein, can be combined include the
following: TNF antagonists, for example, anti-TNF antibodies, D2E7
(PCT Publication No. WO 97/29131; HUMIRA.RTM.), CA2
(REMICADE.RTM.), CDP 571, TNFR-Ig constructs, (p75TNFRIgG
(ENBREL.RTM.) and p55TNFRIgG (Lenercept)) inhibitors and PDE4
inhibitors. Binding proteins of the invention, or antigen binding
portions thereof, can be combined with corticosteroids, for
example, budenoside and dexamethasone. Binding proteins of the
invention, or antigen binding portions thereof, may also be
combined with agents such as sulfasalazine, 5-aminosalicylic acid
and olsalazine, and agents that interfere with synthesis or action
of proinflammatory cytokines such as IL-1, for example, IL-1.beta.
converting enzyme inhibitors and IL-1RA. Binding proteins of the
invention or antigen binding portion thereof may also be used with
T cell signaling inhibitors, for example, tyrosine kinase
inhibitors 6-mercaptopurines. Binding proteins of the invention, or
antigen binding portions thereof, can be combined with IL-11.
Binding proteins of the invention, or antigen binding portions
thereof, can be combined with mesalamine, prednisone, azathioprine,
mercaptopurine, infliximab, methylprednisolone sodium succinate,
diphenoxylate/atrop sulfate, loperamide hydrochloride,
methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,
hydrocodone bitartrate/apap, tetracycline hydrochloride,
fluocinonide, metronidazole, thimerosal/boric acid,
cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine
sulfate, meperidine hydrochloride, midazolam hydrochloride,
oxycodone hcl/acetaminophen, promethazine hydrochloride, sodium
phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil,
propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide
disodium, codeine phosphate/apap, colesevelam hcl, cyanocobalamin,
folic acid, levofloxacin, methylprednisolone, natalizumab and
interferon-gamma.
[0209] Non-limiting examples of therapeutic agents for multiple
sclerosis with which an IL-1.beta. binding protein, or antigen
binding portion, of the invention can be combined include the
following: corticosteroids, prednisolone, methylprednisolone,
azathioprine, cyclophosphamide, cyclosporine, methotrexate,
4-aminopyridine, tizanidine, interferon-.beta.1a (AVONEX.RTM.;
Biogen), interferon-.beta.1b (BETASERON.RTM.; Chiron/Berlex),
interferon .alpha.-n3 (Interferon Sciences/Fujimoto),
interferon-.alpha. (Alfa Wassermann/J&J), interferon
.beta.1A-IF (Serono/Inhale Therapeutics), Peginterferon .alpha. 2b
(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE.RTM.; Teva
Pharmaceutical Industries, Inc.), hyperbaric oxygen, intravenous
immunoglobulin, clabribine, antibodies to or antagonists or
inhibitors of other human cytokines or growth factors and their
receptors, for example, TNF, LT, IL-1.beta., IL-2, IL-6, IL-7,
IL-8, IL-1A, 1L-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF.
Antibodies of the invention, or antigen binding portions thereof,
can be combined with antibodies to cell surface molecules such as
CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. The antibodies of the invention,
or antigen binding portions thereof, may also be combined with
agents, such as FK506, rapamycin, mycophenolate mofetil,
leflunomide, NSAIDs, for example, ibuprofen, phosphodiesterase
inhibitors, adensosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, agents which interfere with
signaling by proinflammatory cytokines such as TNF.alpha. or IL-1
(e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1b
converting enzyme inhibitors, TACE inhibitors, T-cell signaling
inhibitors such as kinase inhibitors, metalloproteinase inhibitors,
sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin
converting enzyme inhibitors, soluble cytokine receptors and
derivatives thereof (e.g., soluble p55 or p75 TNF receptors,
sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g., IL-4,
IL-10, IL-13 and TGF.beta.), COPAXONE.RTM., and caspase inhibitors,
for example inhibitors of caspase-1.
[0210] The IL-1.beta. binding proteins of the invention, or antigen
binding portions thereof, may also be combined with agents, such as
alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,
xaliproden hydrochloride, fampridine, glatiramer acetate,
natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062,
AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine,
CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD
(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,
anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258
(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-.beta.2,
tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4
Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists,
IL-4 agonists.
[0211] Non-limiting examples of therapeutic agents for the
treatment or prevention of angina with which an IL-1.beta. binding
protein, or antigen binding portion thereof, of the invention can
be combined include the following: aspirin, nitroglycerin,
isosorbide mononitrate, metoprolol succinate, atenolol, metoprolol
tartrate, amlodipine besylate, diltiazem hydrochloride, isosorbide
dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium,
potassium chloride, furosemide, simvastatin, verapamil hcl,
digoxin, propranolol hydrochloride, carvedilol, lisinopril,
spironolactone, hydrochlorothiazide, enalapril maleate, nadolol,
ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
and bisoprolol fumarate.
[0212] Non-limiting examples of therapeutic agents for the
treatment or prevention of ankylosing spondylitis with which a
binding protein, or antigen binding portion thereof, of the
invention can be combined include the following: ibuprofen,
diclofenac and misoprostol, naproxen, meloxicam, indomethacin,
diclofenac, celecoxib, rofecoxib, sulfasalazine, methotrexate,
azathioprine, minocyclin, prednisone, etanercept, and
infliximab.
[0213] Non-limiting examples of therapeutic agents for the
treatment or prevention of asthma with which an IL-1.beta. binding
protein, or antigen binding portion thereof, of the invention can
be combined include the following: albuterol,
salmeterol/fluticasone, montelukast sodium, fluticasone propionate,
budesonide, prednisone, salmeterol xinafoate, levalbuterol hcl,
albuterol sulfate/ipratropium, prednisolone sodium phosphate,
triamcinolone acetonide, beclomethasone dipropionate, ipratropium
bromide, azithromycin, pirbuterol acetate, prednisolone,
theophylline anhydrous, methylprednisolone sodium succinate,
clarithromycin, zafirlukast, formoterol fumarate, influenza virus
vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide,
allergy injection, cromolyn sodium, fexofenadine hydrochloride,
flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler
assist device, guaifenesin, dexamethasone sodium phosphate,
moxifloxacin hcl, doxycycline hyclate, guaifenesin/d-methorphan,
p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine
hydrochloride, mometasone furoate, salmeterol xinafoate,
benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine
hcl/pseudoephed, phenylephrine/cod/promethazine,
codeine/promethazine, cefprozil, dexamethasone,
guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone,
nedocromil sodium, terbutaline sulfate, epinephrine,
methylprednisolone, and metaproterenol sulfate.
[0214] Non-limiting examples of therapeutic agents for the
treatment or prevention of COPD with which an IL-1.beta. binding
protein, or antigen binding portion thereof, of the invention can
be combined include the following: albuterol sulfate/ipratropium,
ipratropium bromide, salmeterol/fluticasone, albuterol, salmeterol
xinafoate, fluticasone propionate, prednisone, theophylline
anhydrous, methylprednisolone sodium succinate, montelukast sodium,
budesonide, formoterol fumarate, triamcinolone acetonide,
levofloxacin, guaifenesin, azithromycin, beclomethasone
dipropionate, levalbuterol hcl, flunisolide, ceftriaxone sodium,
amoxicillin trihydrate, gatifloxacin, zafirlukast,
amoxicillin/clavulanate, flunisolide/menthol,
chlorpheniramine/hydrocodone, metaproterenol sulfate,
methylprednisolone, mometasone furoate,
p-ephedrine/cod/chlorphenir, pirbuterol acetate,
p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide,
(R,R)-formoterol, TgAAT, cilomilast, and roflumilast.
[0215] Non-limiting examples of therapeutic agents for the
treatment or prevention of HCV with which an IL-1.beta. binding
protein, or antigen binding portion thereof, of the invention can
be combined include the following: interferon-alpha-2a,
interferon-alpha-2b, interferon-alpha con1, interferon-alpha-n1,
pegylated interferon-alpha-2a, pegylated interferon-alpha-2b,
ribavirin, peginterferon alfa-2b+ ribavirin, ursodeoxycholic acid,
glycyrrhizic acid, thymalfasin, maxamine, VX-497 and any compounds
that are used to treat HCV through intervention with the following
targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES
(internal ribosome entry site).
[0216] Non-limiting examples of therapeutic agents for the
treatment or prevention of idiopathic pulmonary fibrosis with which
a binding protein, or antigen binding portion thereof, of the
invention can be combined include the following: prednisone,
azathioprine, albuterol, colchicine, albuterol sulfate, digoxin,
gamma interferon, methylprednisolone sod succ, lorazepam,
furosemide, lisinopril, nitroglycerin, spironolactone,
cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,
fluticasone propionate, levofloxacin, metaproterenol sulfate,
morphine sulfate, oxycodone HCl, potassium chloride, triamcinolone
acetonide, tacrolimus anhydrous, calcium, interferon-alpha,
methotrexate, mycophenolate mofetil, and interferon-gamma-1b.
[0217] Non-limiting examples of therapeutic agents for the
treatment or prevention of myocardial infarction with which an
IL-1.beta. binding protein, or antigen binding portion thereof, of
the invention can be combined include the following: aspirin,
nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin
sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine
sulfate, metoprolol succinate, warfarin sodium, lisinopril,
isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,
tenecteplase, enalapril maleate, torsemide, retavase, losartan
potassium, quinapril hcl/mag carb, bumetanide, alteplase,
enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate,
diltiazem hydrochloride, captopril, irbesartan, valsartan,
propranolol hydrochloride, fosinopril sodium, lidocaine
hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate,
aminocaproic acid, spironolactone, interferon, sotalol
hydrochloride, potassium chloride, docusate sodium, dobutamine hcl,
alprazolam, pravastatin sodium, atorvastatin calcium, midazolam
hydrochloride, meperidine hydrochloride, isosorbide dinitrate,
epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,
ezetimibe/simvastatin, avasimibe, and cariporide.
[0218] Non-limiting examples of therapeutic agents for the
treatment or prevention of psoriasis with which an IL-1.beta.
binding protein, or antigen binding portion thereof, of the
invention can be combined include the following: calcipotriene,
clobetasol propionate, triamcinolone acetonide, halobetasol
propionate, tazarotene, methotrexate, fluocinonide, betamethasone
diprop augmented, fluocinolone acetonide, acitretin, tar shampoo,
betamethasone valerate, mometasone furoate, ketoconazole,
pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide,
urea, betamethasone, clobetasol propionate/emoll, fluticasone
propionate, azithromycin, hydrocortisone, moisturizing formula,
folic acid, desonide, pimecrolimus, coal tar, diflorasone
diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth
subgal/znox/resor, methylprednisolone acetate, prednisone,
sunscreen, halcinonide, salicylic acid, anthralin, clocortolone
pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic
acid/sulfur, desoximetasone, diazepam, emollient,
fluocinonide/emollient, mineral oil/castor oil/na lact, mineral
oil/peanut oil, petroleum/isopropyl myristate, psoralen, salicylic
acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,
infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,
pimecrolimus, PUVA, UVB, and sulfasalazine.
[0219] Non-limiting examples of therapeutic agents for the
treatment or prevention of psoriatic arthritis with which an
IL-1.beta. binding protein, or antigen binding portion thereof, of
the invention can be combined include the following: methotrexate,
etanercept, rofecoxib, celecoxib, folic acid, sulfasalazine,
naproxen, leflunomide, methylprednisolone acetate, indomethacin,
hydroxychloroquine sulfate, prednisone, sulindac, betamethasone
diprop augmented, infliximab, methotrexate, folate, triamcinolone
acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac
sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone,
tolmetin sodium, calcipotriene, cyclosporine, diclofenac
sodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodium
thiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate
sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, and
efalizumab.
[0220] Non-limiting examples of therapeutic agents for the
treatment or prevention of restenosis with which an IL-1.beta.
binding protein, or antigen binding portion thereof, of the
invention can be combined include the following: sirolimus,
paclitaxel, everolimus, tacrolimus, ABT-578, and acetaminophen.
[0221] Non-limiting examples of therapeutic agents for the
treatment or prevention of sciatica with which an IL-1.beta.
binding protein, or antigen binding portion thereof, of the
invention can be combined include the following: hydrocodone
bitartrate/apap, rofecoxib, cyclobenzaprine hcl,
methylprednisolone, naproxen, ibuprofen, oxycodone
hcl/acetaminophen, celecoxib, valdecoxib, methylprednisolone
acetate, prednisone, codeine phosphate/apap, tramadol
hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine
hydrochloride, diclofenac sodium, gabapentin, dexamethasone,
carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen,
diazepam, nabumetone, oxycodone hcl, tizanidine hcl, diclofenac
sodium/misoprostol, propoxyphene napsylate/apap,
asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,
etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine
phos/asa, morphine sulfate, multivitamins, naproxen sodium,
orphenadrine citrate, and temazepam.
[0222] Non-limiting examples of therapeutic agents for the
treatment or prevention of systemic lupus erythematosis (SLE) with
which an IL-1.beta. binding protein, or an antigen binding portion
thereof, of the invention can be combined include the following:
NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam,
indomethacin, COX2 inhibitors, for example, celecoxib, rofecoxib,
valdecoxib, anti-malarials, for example, hydroxychloroquine,
steroids, for example, prednisone, prednisolone, budenoside,
dexamethasone, cytotoxics, for example, azathioprine,
cyclophosphamide, mycophenolate mofetil, methotrexate, inhibitors
of PDE4 or of purine synthesis inhibitor, for example,
CELLCEPT.RTM.. Binding proteins of the invention, or antigen
binding portions thereof, may also be combined with agents such as
sulfasalazine, 5-aminosalicylic acid, olsalazine, Imuran and agents
which interfere with synthesis, production or action of
proinflammatory cytokines such as IL-1, for example, caspase
inhibitors like IL-1.beta. converting enzyme inhibitors and IL-1ra.
Binding proteins of the invention, or antigen binding portion
thereof, may also be used with T cell signaling inhibitors, for
example, tyrosine kinase inhibitors, or molecules that target T
cell activation molecules, for example, CTLA-4-IgG or anti-B7
family antibodies, and anti-PD-1 family antibodies. Binding
proteins of the invention, or antigen binding portions thereof, can
be combined with IL-11 or anti-cytokine antibodies, for example,
fonotolizumab (anti-IFNg antibody), or anti-receptor receptor
antibodies, for example, anti-IL-6 receptor antibody and antibodies
to B-cell surface molecules. Binding proteins of the invention, or
antigen binding portion thereof, may also be used with LJP 394
(abetimus), agents that deplete or inactivate B-cells, for example,
rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody),
TNF antagonists, for example, anti-TNF antibodies, D2E7 (PCT
Publication No. WO 97/29131; HUMIRA.RTM.), CA2 (REMICADE.RTM.), CDP
571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL.RTM.) and p55TNFRIgG
(Lenercept)).
[0223] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an IL-1.beta. binding protein, or antigen
binding portion thereof, 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 a binding protein, or
antigen binding portion thereof, described herein may be determined
by a person skilled in the art and may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody, or antigen binding portion
thereof, 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 antigen binding portion
thereof, 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.
[0224] 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.
[0225] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of IL-1.beta. binding protein, or
antigen binding portion thereof, of the invention is about 0.1 to
about 20 mg/kg, about 1 to about 10 mg/kg. Dosage values may vary
with the type and severity of the condition to be alleviated. 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. Dosage ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition.
[0226] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the compositions
and the methods of the invention described herein are obvious and
may be made using suitable equivalents without departing from the
scope of the invention or the embodiments disclosed herein. The
present invention will be more clearly understood by reference to
the following examples, which are included for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
Generation of Anti-Human IL-1.beta. Monoclonal Antibodies
[0227] Mouse anti-human IL-1.beta. monoclonal antibodies are
obtained as follows:
Example 1.1
Immunization of Mice with Human IL-1.beta. Antigen
[0228] Twenty micrograms of recombinant purified human IL-1.beta.
(R&D Systems, Minneapolis, Minn., USA) mixed with complete
Freund's adjuvant or Immunoeasy adjuvant (Qiagen, Valencia, Calif.)
is injected subcutaneously into five 6-8 week-old Balb/C, five
C57B/6 mice, and five AJ mice on Day 1. On days 24, 38, and 49,
twenty micrograms of recombinant purified human IL-1.beta. variant
mixed with incomplete Freund's adjuvant or Immunoeasy adjuvant is
injected subcutaneously into the same mice. On day 84 or day 112 or
day 144, mice are injected intravenously with 1 .mu.g recombinant
purified human IL-1.beta. variant.
Example 1.2
Generation of Hybridoma
[0229] Splenocytes obtained from the immunized mice described in
Example 1.1 are fused with SP2/O-Ag-14 cells at a ratio of 5:1
according to the established method described in Kohler, G. and
Milstein (1975) Nature 256:495 to generate hybridomas. Fusion
products are plated in selection media containing azaserine and
hypoxanthine in 96-well plates at a density of 2.5.times.10.sup.6
spleen cells per well. Seven to ten days post fusion, macroscopic
hybridoma colonies are observed. Supernatant from each well
containing hybridoma colonies is tested by ELISA for the presence
of antibody to IL-1.beta. (as described in Example 3.1).
Supernatants displaying IL-1.beta. specific activity are then
tested for the ability to neutralize IL-1.beta. in the MRC-5
bioassay for IL-8 (as described in Example 3.2).
Example 1.3
Identification and Characterization of Anti Human IL-1.beta.
Monoclonal Antibodies
[0230] Hybridomas producing antibodies that bound IL-1.beta. and
are capable of binding IL-1.beta. specifically and particularly
those with IC.sub.50 values in the MRC-5 bioassay of 5 nM or less
than 5 nM are scaled up and cloned by limiting dilution.
[0231] Hybridoma cells are expanded into media containing 10% low
IgG fetal bovine serum (Hyclone #SH30151, Logan, Utah). On average,
250 mL of each hybridoma supernatant (derived from a clonal
population) is harvested, concentrated and purified by protein A
affinity chromatography by standard methods. The ability of
purified mAbs to inhibit IL-1.beta. activity is determined using
the MRC-5 bioassay as described in Example 3.2.
Example 1.4
Determination of the Amino Acid Sequence of the Variable Region for
Each Murine Anti-Human IL-1.beta. Monoclonal Antibody
[0232] For each amino acid sequence determination, approximately
10.times.10.sup.6 hybridoma cells are isolated by centrifugation
and processed to isolate total RNA with Trizol (Gibco
BRL/Invitrogen, Carlsbad, Calif.) following manufacturer's
instructions. Total RNA is subjected to first strand DNA synthesis
using the SuperScript First-Strand Synthesis System (Invitrogen,
Carlsbad, Calif.) per the manufacturer's instructions. Oligo(dT) is
used to prime first-strand synthesis to select for poly(A).sup.+
RNA. The first-strand cDNA product is then amplified by PCR with
primers designed for amplification of murine immunoglobulin
variable regions (Ig-Primer Sets, Novagen, Madison, Wis.). PCR
products are resolved on an agarose gel, excised, purified, and
then subcloned with the TOPO Cloning kit into pCR2.1-TOPO vector
(Invitrogen, Carlsbad, Calif.) and transformed into TOP10
chemically competent E. coli (Invitrogen, Carlsbad, Calif.). Colony
PCR is performed on the transformants to identify clones containing
insert. Plasmid DNA is isolated from clones containing insert using
a QIAprep Miniprep kit (Qiagen, Valencia, Calif.). Inserts in the
plasmids are sequenced on both strands to determine the variable
heavy or variable light chain DNA sequences using M13 forward and
M13 reverse primers (Fermentas Life Sciences, Hanover Md.).
Variable heavy and variable light chain sequences of the
anti-IL-1.beta. monoclonal antibodies are shown in Table 5.
Example 2
Recombinant Anti-Human IL-1.beta. Antibodies
Example 2.1
Construction and Expression of Recombinant Chimeric Anti-Human
IL-1.beta. Antibodies
[0233] The DNA encoding the heavy chain constant region of murine
anti-human IL-1.beta. monoclonal antibody 1B12.4H4 was replaced by
a cDNA fragment encoding the human IgG1 constant region containing
2 hinge-region amino acid mutations by homologous recombination in
bacteria. These mutations are a leucine to alanine change at
position 234 (EU numbering) and a leucine to alanine change at
position 235 (Lund et al. (1991) J. Immunol. 147:2657). The light
chain constant region of each of these antibodies was replaced by a
human kappa constant region. Full-length chimeric antibodies were
transiently expressed in HEK 293-6E cells by co-transfection of
chimeric heavy and light chain cDNAs ligated into the pHybE
expression plasmid (US Patent Publication No. US 20090239259). Cell
supernatants containing recombinant chimeric antibody were purified
by Protein A Sepharose chromatography and bound antibody was eluted
by addition of acid buffer. Antibodies were neutralized and
dialyzed into PBS.
[0234] The heavy chain cDNA encoding chimeric 1B12.4H4 was
co-transfected with the 1B12.4H4 chimeric light chain cDNA (both
ligated in the pHybE vector) into HEK 293-6E cells. Cell
supernatant containing recombinant chimeric antibody was purified
by Protein A Sepharose chromatography and bound antibody was eluted
by addition of acid buffer. Antibodies were neutralized and
dialyzed into PBS. The purified chimeric anti-human IL-1.beta.
monoclonal antibodies were then tested for their ability to inhibit
the IL-1.beta. induced production of IL-8 by MRC-5 cells as
described in Example 3.2.
Example 2.2
Construction and Expression of Humanized Anti-Human IL-1.beta.
Antibodies
Example 2.2.1
Selection of Human Antibody Frameworks
[0235] The canonical structures of the VH and VL CDRs were
determined according to the method of Huang et al. (2005) Methods
36:35-42. Based on the canonical structures, the appropriate
acceptor human VH framework sequences were 3-13, 3-53, 3-66, 4-34,
and 4-59 and the appropriate acceptor human VL framework sequences
included Vk1, some Vk3, Vk5, and Vk6 subgroups. hJH4 was selected
as the acceptor human FR4 sequence based on an alignment of amino
acids 109-122 of the 1B12VH region with hJH1-6 sequences.
[0236] Four additional VH sequences (1B12VHx, -xx, -xxx, and -xxxx)
were created by gradually replacing CDR or framework residues with
`X`. 1B12VHs was the VH sequence with D and J regions removed. The
five sequences were aligned with human VH sequences in the Align X
program of Vector NTI.RTM. software (Invitrogen, Carlsbad, Calif.).
Focusing on overall framework or only specific residues important
for loop conformation and VH/VL interface, the VH4-59 had the best
overall homology to the 1B12 VH sequence. This framework was
selected as the accepter human germline framework for grafting 1B12
VH CDR sequences. The VH3-53 was selected as the alternative
acceptor human framework from a different VH family. VH4 sequences
were aligned to identify framework residues in VH4-59 that could be
changed into VH4 consensus sequence to minimize immunogenicity
potential of the humanized sequence. Sequence alignment of VH4-59
showed there was no need to introduce framework residue changes to
bring VH4-59 to a VH4 consensus sequence. Sequence alignment showed
that the VH4-59 sequence to be used as an acceptor framework was
the dominant consensus. Similarly, all VH3 sequences were aligned
to identify framework residues in VH3-53 that could be changed into
VH3 consensus sequence to minimize immunogenicity potential. Two
VH3 consensus changes (I12V and V29F) were identified. The
alignment also showed that the VH3-53 sequence to be used as an
acceptor framework was the first sequence from the three known
VH3-53 polymorphisms. Sequences in set one having from one to eight
out of the nine proposed back-mutations in any combinations with or
without the Q1E change can be made to produce additional humanized
1B12 VH sequences with less immunogenicity potential or better
overall identity to naturally occurring human VH sequences from the
VH4-59 germline sequence.
[0237] To determine the prevalence of the proposed back-mutations
in human antibodies originated from VH4-59, human VH sequences
derived from VH4-59 were downloaded from the NCBI IgBlast database
into a batch fasta file, aligned by ClustalW, and visualized by
logobar. The output .eps file was edited by Adobe Illustrator to
remove gaps, signal peptide, most of CDR3, and constant region
sequences. This analysis was useful for understanding whether the
proposed back-mutations and the mouse VH CDR residues were
represented in 825 natural human antibodies that could be
originated from VH4-59. Of the nine proposed (G27F, I29L, I48L,
V67L, V71K, T73N, N76S, F78V, and R94K) back-mutations, G27F, I29L,
V67L, N76S, F78V, and R94K were observed in more than 1% of these
sequences.
[0238] Sequences in set two having from one to nine out of the ten
proposed back-mutations in any combinations having zero to two out
of the two I12V and V29F VH3 framework consensus changes can be
made to produce additional humanized 1B12 VH sequences with less
immunogenicity potential or better overall identity to naturally
occurring human VH sequences from the VH3-53 germline sequence.
Human VH sequences derived from VH3-53 were downloaded from the
NCBI IgBlast database into a batch fasta file, aligned by ClustalW,
and visualized by logobar. The output .eps file was edited by Adobe
Illustrator to remove gaps, signal peptide, and constant region
sequences. This analysis was useful for understanding whether the
proposed ten back-mutations and the mouse VH CDR residues were
represented in 174 natural human antibodies that could be
originated from VH3-53. Of the ten proposed (A24V, F29L, V371,
V48L, S49G, F67L, R71K, N76S, L78V, and R94K) back-mutations, A24V,
F29L, R71K, N76S, L78V, and R94K were observed in more than 1% of
these sequences.
[0239] Residues supporting loop structures and VH/VL interface in
VL sequences were summarized in PCT Publication No. WO2008021156.
Four additional VL sequences (1B12VLx, -xx, -xxx, and -xxx) were
created by gradually replacing CDR or framework residues with `X`.
1B12VLs is the VL sequence with J region removed. The five
sequences were aligned with human Vk sequences in the Align X
program of Vector NTI.RTM. software. Only human Vk germline
sequences having 2-1-1 canonical CDR structures were considered.
The human Vk germline 1-33/018 was selected as the acceptor
framework for 1B12.4H4 VL humanization. The human Vk germline
6D41/A14 was selected as the back-up acceptor framework for
humanization from a different subgroup. All human Vk1 germline
sequences were aligned to identify potential framework residues in
1-33/018 that should be changed into Vk1 consensus to minimize
immunogenicity potential. Two Vk1 consensus changes (F73L and 183F)
were identified. These changes minimized the immunogenicity
potential of the humanized 1B12 VL sequence. The same analysis was
not done for the 6D41/A14 as there were only two different germline
sequences in the Vk6 subgroup.
[0240] Additional sequences of set one containing one to six out of
the seven proposed back-mutations (I2T, M4V, A43P, Y49S, G64S, D1E,
and Q3T) in any combinations with or without the 183F Vk1 consensus
change can be made to achieve better IgG function, less
immunogenicity potential, or better overall identity to naturally
occurring human VL sequences from the 018 germline sequence.
Additional sequences of set two having one to six out of the seven
proposed back-mutations (V2T, M4V, A43P, K49S, G64S, D1E, and V3T)
in any combinations can be made to achieve better IgG function,
less immunogenicity potential, or better overall homology to
naturally occurring human VL sequences from the 6D41/A14 germline
sequence.
[0241] Human Vk sequences derived from 1-33/018 were downloaded
from the NCBI IgBlast database into one batch fasta file, aligned
by ClustalW, and visualized by logobar. The output .eps file was
edited by Adobe Illustrator to remove gaps, signal peptide, and
constant region sequences. This analysis was useful for
understanding whether the proposed back-mutations and the mouse VH
CDR residues were represented in 260 natural human antibodies that
could be originated from 1-33/018. Of the seven proposed
back-mutations, D1E and Y49S were observed in more than 1% of these
sequences.
Example 2.2.2
Humanization of Anti-Human IL-1.beta. Monoclonal Antibody
1B12.4H4
[0242] The heavy chain CDR sequences from the anti-IL-1.beta.
antibody 1B12.4H4 described in Table 5 were grafted in silico onto
two human frameworks. The first set comprises the mAbs h1B12VH.1z,
h1B12VH.1, and h1B12VH.1a. h1B12VH.1z is a CDR-grafted humanized
1B12 VH containing VH4-59 and JH4 framework sequences. h1B12VH.1 is
a humanized design further incorporating a Q1E framework change to
prevent N-terminal pyroglutamate formation. h1B12VH.1a is a
humanized design containing the Q1E change and all possible
framework back-mutations G27F, I29L, I48L, V67L, V71K, T73N, N76S,
F78V, and R94K.
[0243] The second set comprises the mAbs h1B12VH.2z, h1B12VH.2, and
h1B12VH.2a. h1B12VH.2z is a CDR-grafted humanized 1B12 VH
containing VH3-53 and hJH4 framework sequences. h1B12VH.2 is a
humanized design incorporating I12V and V29F VH3 framework
consensus changes. h1B12VH.2a is a humanized design containing the
VH3 framework consensus changes and all possible framework
back-mutations A24V, F29L (F is a VH3 consensus sequence), V37I,
V48L, S49G, F67L, R71K, N76S, L78V, and R94K. No N-linked
glycosylation pattern (N-{P}-S/T) was found in these proposed
constructs. Not all back-mutations may be needed. The
back-mutations I48L, V71K, and T73N in set 1 and F29L, V37I, V48L,
S49G, and F67L in set 2 are not represented in human antibodies.
Some back-mutations could be removed during subsequent affinity
maturation process if so desired.
[0244] The light chain CDR sequences from the anti-IL-1.beta.
antibody 1B12.4H4 described in Table 5 were grafted in silico onto
two human frameworks. The first set comprises the mAbs h1B12VL.1z,
h1B12VL.1, h1B12VL.1a, and h1B12VL.1b. h1B12VL.1z is a direct
CDR-grafted humanized 1B12 VL containing O18 and Jk2 framework
sequences. h1B12VL.1 is a humanized design incorporating 183F Vk1
framework consensus change. h1B12VL.1a is a humanized design
containing the consensus change and 5 framework back-mutations
(I2T, M4V, A43P, Y49S, G64S). h1B12VL.1b is a humanized design
containing the consensus changes and 7 framework back-mutations (5
above and 2 additional N-terminal back-mutations D1E and Q3T). The
second set comprises the mAbs h1B12VL.2, h1B12VL.2a, and
h1B12VL.2b. h1B12VL.2 is a direct CDR-grafted humanized 1B12 VL
containing A14 and Jk2 framework sequences. h1B12VL.2a is a
humanized design containing 5 framework back-mutation (V2T, M4V,
A43P, K49S, G64S). h1B12VL.2b is a humanized design containing 7
framework back-mutations (5 above and 2 additional N-terminal
back-mutations D1E and V3T). No N-linked glycosylation pattern
(N-{P}-S/T) was found in the proposed constructs. Not all
back-mutations may be needed. The back-mutations I2T, Q3T, M4V,
A43P, and G64S in version 1 are not represented in human
antibodies. There are very few human antibodies originated from
6D41/A14 germline sequence. Some back-mutations could be removed
during subsequent affinity maturation process if so desired.
[0245] Table 6 is a list of amino acid sequences of VH and VL
regions of humanized anti-hIL-1.beta. antibodies of the
invention.
TABLE-US-00006 TABLE 6 List Of Amino Acid Sequences Of Humanized
1B12 VH/VL Variants SEQ ID Sequence No. Protein region
123456789012345678901234567890 28 h1B12AH4VH.1z
QVQLQESGPGLVKPSETLSLTCTVSGGSIS DYGVSWIRQPPGKGLEWIGLIWGGGDTYYN
SPLKSRVTISVDTSKNQFSLKLSSVTAADT AVYYCARQRTLWGYDLYGMDYWGQGTLVTV SS 29
h1B12.4H4VH.1 EVQLQESGPGLVKPSETLSLTCTVSGGSIS
DYGVSWIRQPPGKGLEWIGLIWGGGDTYYN SPLKSRVTISVDTSKNQFSLKLSSVTAADT
AVYYCARQRTLWGYDLYGMDYWGQGTLVTV SS 30 h1B12.4H4VH.1a
EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN
SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 31
h1B12.4H4VH.2z EVQLVESGGGLIQPGGSLRLSCAASGFTVS
DYGVSWVRQAPGKGLEWVSLIWGGGDTYYN SPLKSRFTISRDNSKNTLYLQMNSLRAEDT
AVYYCARQRTLWGYDLYGMDYWGQGTLVTV SS 32 h1B12.4H4VH.2
EVQLVESGGGLVQPGGSLRLSCAASGFTFS DYGVSWVRQAPGKGLEWVSLIWGGGDTYYN
SPLKSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARQRTLWGYDLYGMDYWGQGTLVTV SS 33
h1B12AH4VH.2a EVQLVESGGGLVQPGGSLRLSCAVSGFTLS
DYGVSWIRQAPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSTVYLQMNSLRAEDT
AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 34 h1B12.4H4VL.1
DIQMTQSPSSLSASVGDRVTITCITSTDID VDMNWYQQKPGKAPKLLIYQGNTLRPGVPS
RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIK 35 h1B12.4H4VL.1z
DIQMTQSPSSLSASVGDRVTITCITSTDID VDMNWYQQKPGKAPKLLIYQGNTLRPGVPS
RFSGSGSGTDFTFTISSLQPEDIATYYCLQ SDNLPLTFGQGTKLEIK 36 h1B12AH4VL.1a
DTQVTQSASSLSASVGDRVTITCITSTDID VDMNWYQQKPGKPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIK 37 h1B12.4H4VL.1b
ETTVTQSPSSLSASVGDRVTITCITSTDID VDMNWYQQKPGKPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIK 38 h1B12.4H4VL.2
DVVMTQSPAFLSVTPGEKVTITCITSTDID VDMNWYQQKPDQAPKLLIKQGNTLRPGVPS
RFSGSGSGTDFTFTISSLEAEDAATYYCLQ SDNLPLTFGQGTKLEIK 39 h1B12.4H4VL.2a
DTVVTQSPAFLSVTPGEKVTITCITSTDID VDMNWYQQKPDQPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLEAEDAATYYCLQ SDNLPLTFGQGTKLEIK 40 h1B12.4H4VL.2b
ETTVTQSPAFLSVTPGEKVTITCITSTDID VDMNWYQQKPDQPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLEAEDAATYYCLQ SDNLPLTFGQGTKLEIK
Example 2.2.3
Construction of Humanized Antibodies
[0246] The sequences can be used to synthesize nucleic acids using
standard DNA synthesis or amplification technologies and assembling
the desired antibody fragments into expression vectors, using
standard recombinant DNA technology, for expression in cells. For
example, nucleic acid codons are determined from amino acids
sequences and oligonucleotide DNA is synthesized by Blue Heron
Biotechnology, Inc. (www.blueheronbio.com) Bothell, Wash. USA. The
oligonucleotides are assembled into 300-2,000 base pair
double-stranded DNA fragments, cloned into a plasmid vector and
sequence-verified. Cloned fragments are assembled using an
enzymatic process to yield the complete gene and subcloned into an
expression vector. (See U.S. Pat. Nos. 7,306,914; 7,297,541;
7,279,159; 7,150,969; 20080115243; 20080102475; 20080081379;
20080075690; 20080063780; 20080050506; 20080038777; 20080022422;
20070289033; 20070287170; 20070254338; 20070243194; 20070225227;
20070207171; 20070150976; 20070135620; 20070128190; 20070104722;
20070092484; 20070037196; 20070028321; 20060172404; 20060162026;
20060153791; 20030215458; and 20030157643).
[0247] For example, in silico constructed humanized antibodies
described above can be inserted into the multiple cloning site in a
pHybE vector (US Patent Publication No. US 2009/0239259). Bacterial
colonies are isolated and plasmid DNA extracted; cDNA inserts are
sequenced in their entirety. Correct humanized heavy and light
chains corresponding to each antibody are co-transfected into HEK
293-6E cells to transiently produce full-length humanized
anti-human IL-1.beta. antibodies. pHybE vectors containing the
heavy chain grafted cDNA and the light chain grafted cDNA were
co-transfected into HEK 293-6E cells. Cell supernatants containing
recombinant chimeric antibody are purified by Protein A Sepharose
chromatography and bound antibody is eluted by addition of acid
buffer. Antibodies are neutralized and dialyzed into PBS. Humanized
antibodies are described in Table 7.
[0248] The different combinations of humanized 1B12 antibody based
on the VH and VL shuffling are listed in Table 7.
TABLE-US-00007 TABLE 7 Combinations Of Humanized 1B12 Antibody
Based On The VH And VL Shuffling 1B12 h1B12 h1B12 h1B12 h1B12 h1B12
h1B12 h1B12 VL VL.1z VL.1 VL.1a VL.1b VL.2 VL.2a VL.2b 1B12VH
h1B12VH.1z h1B12VH.1 h1B12VH.1a 1 2 3 4 h1B12VH.2z h1B12VH.2
h1B12VH.2a 5 6 7 8
[0249] Table 8 provides humanized variable regions of the murine
monoclonal antibody 1B12 that were cloned into IgG expression
vectors for functional characterization. CDRs are shown in
bold.
TABLE-US-00008 TABLE 8 Humanized Variable Regions Of The Murine
Monoclonal Antibody 1B12 SEQ ID Protein Sequence No. region
123456789012345678901234567890 30 1B12.1
EVQLQESGPGLVKPSETLSLTCTVSGFSLS VH DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN
SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41
1B12.1 CDR H1 Residues DYGVS VH 31-35 of SEQ ID NO: 26 42 1B12.1
CDR H2 Residues LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43
1B12.1 CDR H3 Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 36
1B12.1 DTQVTQSPSSLSASVGDRVTITCITSTDID VL
VDMNWYQQKPGKPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLQPEDFATYYCLQ
SDNLPLTFGQGTKLEIK 44 1B12.1 CDR L1 Residues ITSTDIDVDMN VL 24-34 of
SEQ ID NO: 27 45 1B12.1 CDR L2 Residues QGNTLRP VL 50-56 of SEQ ID
NO: 27 46 1B12.1 CDR L3 Residues LQSDNLPLT VL 89-97 of SEQ ID NO:
27 30 1B12.2 EVQLQESGPGLVKPSETLSLTCTVSGFSLS VH
DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT
AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41 1B12.2 CDR H1 Residues DYGVS
VH 31-35 of SEQ ID NO: 26 42 1B12.2 CDR H2 Residues
LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43 1B12.2 CDR H3
Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 37 1B12.2
ETTVTQSPSSLSASVGDRVTITCITSTDID VL VDMNWYQQKPGKPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIK 44 1B12.2 CDR L1
Residues ITSTDIDVDMN VL 24-34 of SEQ ID NO: 27 45 1B12.2 CDR L2
Residues QGNTLRP VL 50-56 of SEQ ID NO: 27 46 1B12.2 CDR L3
Residues LQSDNLPLT VL 89-97 of SEQ ID NO: 27 30 1B12.3
EVQLQESGPGLVKPSETLSLTCTVSGFSLS VH DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN
SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41
1B12.3 CDR H1 Residues DYGVS VH 31-35 of SEQ ID NO: 26 42 1B12.3
CDR H2 Residues LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43
1B12.3 CDR H3 Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 39
1B12.3 DTVVTQSPAFLSVTPGEKVTITCITSTDID VL
VDMNWYQQKPDQPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLEAEDAATYYCLQ
SDNLPLTFGQGTKLEIK 44 1B12.3 CDR L1 Residues ITSTDIDVDMN VL 24-34 of
SEQ ID NO: 27 45 1B12.3 CDR L2 Residues QGNTLRP VL 50-56 of SEQ ID
NO: 27 46 1B12.3 CDR L3 Residues LQSDNLPLT VL 89-97 of SEQ ID NO:
27 30 1B12.4 EVQLQESGPGLVKPSETLSLTCTVSGFSLS VH
DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT
AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41 1B12.4 CDR H1 Residues DYGVS
VH 31-35 of SEQ ID NO: 26 42 1B12.4 CDR H2 Residues
LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43 1B12.4 CDR H3
Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 40 1B12.4
ETTVTQSPAFLSVTPGEKVTITCITSTDID VL VDMNWYQQKPDQPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLEAEDAATYYCLQ SDNLPLTFGQGTKLEIK 44 1B12.4 CDR L1
Residues ITSTDIDVDMN VL 24-34 of SEQ ID NO: 27 45 1B12.4 CDR L2
Residues QGNTLRP VL 50-56 of SEQ ID NO: 27 46 1B12.4 CDR L3
Residues LQSDNLPLT VL 89-97 of SEQ ID NO: 27 33 1B12.5
EVQLVESGGGLVQPGGSLRLSCAVSGFTLS VH DYGVSWIRQAPGKGLEWLGLIWGGGDTYYN
SPLKSRLTISKDNSKSTVYLQMNSLRAEDT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41
1B12.5 CDR H1 Residues DYGVS VH 31-35 of SEQ ID NO: 26 42 1B12.5
CDR H2 Residues LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43
1B12.5 CDR H3 Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 36
1B12.5 DTQVTQSPSSLSASVGDRVTITCITSTDID VL
VDMNWYQQKPGKPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLQPEDFATYYCLQ
SDNLPLTFGQGTKLEIK 44 1B12.5 CDR L1 Residues ITSTDIDVDMN VL 24-34 of
SEQ ID NO: 27 45 1B12.5 CDR L2 Residues QGNTLRP VL 50-56 of SEQ ID
NO: 27 46 1B12.5 CDR L3 Residues LQSDNLPLT VL 89-97 of SEQ ID NO:
27 33 1B12.6 EVQLVESGGGLVQPGGSLRLSCAVSGFTLS VH
DYGVSWIRQAPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSTVYLQMNSLRAEDT
AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41 1B12.6 CDR H1 Residues DYGVS
VH 31-35 of SEQ ID NO: 26 42 1B12.6 CDR H2 Residues
LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43 1B12.6 CDR H3
Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 37 1B12.6
ETTVTQSPSSLSASVGDRVTITCITSTDID VL VDMNWYQQKPGKPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIK 44 1B12.6 CDR L1
Residues ITSTDIDVDMN VL 24-34 of SEQ ID NO: 27 45 1B12.6 CDR L2
Residues QGNTLRP VL 50-56 of SEQ ID NO: 27 46 1B12.6 CDR L3
Residues LQSDNLPLT VL 89-97 of
SEQ ID NO: 27 33 1B12.7 EVQLVESGGGLVQPGGSLRLSCAVSGFTLS VH
DYGVSWIRQAPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSTVYLQMNSLRAEDT
AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41 1B12.7 CDR H1 Residues DYGVS
VH 31-35 of SEQ ID NO: 26 42 1B12.7 CDR H2 Residues
LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43 1B12.7 CDR H3
Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 39 1B12.7
DTVVTQSPAFLSVTPGEKVTITCITSTDID VL VDMNWYQQKPDQPPKLLISQGNTLRPGVPS
RFSSSGSGTDFTFTISSLEAEDAATYYCLQ SDNLPLTFGQGTKLEIK 44 1B12.7 CDR L1
Residues ITSTDIDVDMN VL 24-34 of SEQ ID NO: 27 45 1B12.7 CDR L2
Residues QGNTLRP VL 50-56 of SEQ ID NO: 27 46 1B12.7 CDR L3
Residues LQSDNLPLT VL 89-97 of SEQ ID NO: 27 33 1B12.8
EVQLVESGGGLVQPGGSLRLSCAVSGFTLS VH DYGVSWIRQAPGKGLEWLGLIWGGGDTYYN
SPLKSRLTISKDNSKSTVYLQMNSLRAEDT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 41
1B12.8 CDR H1 Residues DYGVS VH 31-35 of SEQ ID NO: 26 42 1B12.8
CDR H2 Residues LIWGGGDTYYNSPLKS VH 50-65 of SEQ ID NO: 26 43
1B12.8 CDR H3 Residues QRTLWGYDLYGMDY VH 98-111 of SEQ ID NO: 26 40
1B12.8 ETTVTQSPAFLSVTPGEKVTITCITSTDID VL
VDMNWYQQKPDQPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLEAEDAATYYCLQ
SDNLPLTFGQGTKLEIK 44 1B12.8 CDR L1 Residues ITSTDIDVDMN VL 24-34 of
SEQ ID NO: 27 45 1B12.8 CDR L2 Residues QGNTLRP VL 50-56 of SEQ ID
NO: 27 46 1B12.8 CDR L3 Residues LQSDNLPLT VL 89-97 of SEQ ID NO:
27
Example 3
Functional Characterization of Human IL-1.beta. Antibodies
Example 3.1
IL-1.beta. Enzyme-Linked Immunosorbent Assay Protocol
[0250] To determine if anti-IL-1.beta. mAbs bind to human
IL-1.beta., ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) were
incubated overnight at 4.degree. C. with anti-human Fc antibody
diluted in Pierce Coat buffer at 2 .mu.g/ml (Jackson
Immunoresearch, West Grove, Pa.). Plates were washed five times in
washing buffer (PBS containing 0.05% Tween 20), and blocked for 1
hour at 25.degree. C. with 200 .mu.l per well superblock blocking
buffer (Thermo scientific, #37515). Blocking buffer was removed by
tapping plates, and 2 .mu.g/ml of each antibody in PBS containing
10% superblock, 0.5% tween-20 was added to the wells at 100 .mu.l
per well and incubated at 25.degree. C. for 1 hour. The wells were
washed five times in 1.times.PBST, and 1 .mu.g/ml biotinylated
antigen was titrated at 1:6 serial dilutions (for a range of .mu.g
to pg in PBS containing 10% superblock, 0.05% tween 20). Each
dilution of antigen was then added to the plates and incubated for
1 hour at 25.degree. C. The wells were washed five times in
1.times.PBST and incubated for 1 hour at 25.degree. C. with polyHRP
streptavidin (KPL #474-3000, Gaithersburg, Md.). The wells were
washed five times in 1.times.PBST, and 100 .mu.l of ULTRA-TMB ELISA
(Pierce, Rockford, Ill.) were added per well. Following color
development the reaction was stopped with 1N HCL and absorbance at
450 nM was measured. The results are shown in Table 9, and the
numerical value indicating binding of human anti-IL-1.beta.
antibodies to human IL-1.beta..
TABLE-US-00009 TABLE 9 Binding Of Humanized Antibodies To Human
IL-1.beta. by ELISA Anti-IL-1.beta. mAb EC50 in hIL-1.beta. ELISA
(pM) 1B12.1 39.9 1B12.2 37.6 1B12.3 43.8 1B12.4 37.8 1B12.5 48.8
1B12.6 45.9 1B12.7 58.2 1B12.8 47.9
Example 3.2
Neutralizing Potency of Humanized IL-1.beta. Antibodies
[0251] To examine the functional activity of the anti-human
IL-1.beta. antibodies in the invention, the antibodies were used in
the MRC-5 assay that measures the ability of the antibody to
inhibit IL-1.beta. activity. The MRC-5 cell line is a human lung
fibroblast cell line that produces IL-8 in response to human
IL-1.beta. in a dose-dependent manner. MRC-5 cells were originally
obtained from ATCC and subcultured in 10% FBS complete MEM and
grown at 37.degree. C. in a 5% CO.sub.2 incubator. To determine an
antibody's neutralizing potency against IL-1.beta., antibodies (50
ul) were added to a 96 well plate (1.times.10.sup.-7 to
1.times.10.sup.-15 M final concentration range) and pre-incubated
with 50 ul of human IL-1.beta. (50 pg/mL final concentration) for 1
hr at 37.degree. C., 5% CO.sub.2. Antigen antibody complexes (100
uL) were then added to MRC-5 cells (plated 24 hours previously at a
concentration of 1E5/ml at 100 ul cells/well). Assay plates were
incubated overnight at 37.degree. C. in a 5% CO.sub.2 incubator.
Antibody potency was determined by its ability to inhibit IL-8
production. Human IL-8 production was measured by a
chemiluminescence-based assay. Table 10 summarizes antibody
potencies to human IL-1.beta..
TABLE-US-00010 TABLE 10 Neutralizing Potency Of Humanized
IL-1.beta. Antibodies Anti-IL-1.beta. mAb Potency on Human
IL-1.beta. IC.sub.50 (pM) 1B12.1 151 1B12.2 146 1B12.3 303 1B12.4
597 1B12.5 319 1B12.6 378 1B12.7 581 1B12.8 484
Example 3.3
Affinity Measurement of IL-1.beta. Antibodies by Surface Plasmon
Resonance
[0252] The BIACORE.TM. assay (Biacore, Inc., Piscataway, N.J.)
determines the affinity of antibodies with kinetic measurements of
on-, off-rate constants. Binding of antibodies to recombinant
purified human IL-1.beta. was determined by surface plasmon
resonance-based measurements with a Biacore.TM. 3000 instrument
(Biacore.TM. AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES
[pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at
25.degree. C. All chemicals were obtained from Biacore.RTM. AB
(Uppsala, Sweden) unless otherwise stated. Approximately 5,000 RU
of goat anti-mouse IgG, (Fc.gamma.), fragment specific polyclonal
antibody (Pierce Biotechnology Inc, Rockford, Ill.) diluted in 10
mM sodium acetate (pH 4.5) was directly immobilized across a CM5
research grade biosensor chip using a standard amine coupling kit
according to manufacturer's instructions and procedures at 25
.mu.g/ml. Unreacted moieties on the biosensor surface were blocked
with ethanolamine. Modified carboxymethyl dextran surface in
flowcell 2 and 4 was used as a reaction surface. Unmodified
carboxymethyl dextran without goat anti-mouse IgG in flow cell 1
and 3 was used as the reference surface. For kinetic analysis, rate
equations derived from the 1:1 Langmuir binding model were fitted
simultaneously to association and dissociation phases of all eight
injections (using global fit analysis) with the use of
Biaevaluation 4.0.1 software. Purified antibodies were diluted in
HEPES-buffered saline for capture across goat anti-mouse IgG
specific reaction surfaces. Mouse antibodies to be captured as a
ligand (25 .mu.g/ml) were injected over reaction matrices at a flow
rate of 5 .mu.l/minute. The association and dissociation rate
constants, k.sub.on (unit M.sup.-1s.sup.-1) and k.sub.off (unit
s.sup.-1) were determined under a continuous flow rate of 25
.mu.l/minute. Rate constants were derived by making kinetic binding
measurements at ten different antigen concentrations ranging from
10-200 nM. The equilibrium dissociation constant (unit M) of the
reaction between antibodies and the target antigen was then
calculated from the kinetic rate constants by the following
formula: K.sub.D=k.sub.off/k.sub.on. Binding is recorded as a
function of time and kinetic rate constants are calculated. In this
assay, on-rates as fast as 10.sup.6M.sup.-1s.sup.-1 and off-rates
as slow as 10.sup.-6 s.sup.-1 can be measured. Table 11 shows the
affinity measurements for human anti-IL-1.beta. antibodies.
TABLE-US-00011 TABLE 11 Affinity of Humanized Antibodies To Human
IL-1.beta. By Biacore K.sub.a (1/Ms) K.sub.d (1/s) K.sub.D (M)
1B12.1 5.55E+06 1.03E-03 1.86E-10 1B12.2 6.13E+06 0.90E-03 1.47E-10
1B12.3 5.73E+06 1.18E-03 2.06E-10 1B12.4 6.01E+06 0.97E-03 1.61E-10
1B12.5 6.24E+06 1.44E-03 2.31E-10 1B12.6 6.20E+06 1.25E-03 2.02E-10
1B12.7 5.76E+06 1.64E-03 2.84E-10 1B12.8 5.94E+06 1.37E-03
2.30E-10
[0253] The present invention incorporates by reference in their
entirety techniques well known in the field of molecular biology.
These techniques include, but are not limited to, techniques
described in the following publications: [0254] Ausubel et al.
eds., Short Protocols In Molecular Biology (4th Ed. 1999) John
Wiley & Sons, NY (ISBN 0-471-32938-X). [0255] Lu and Weiner
eds., Cloning and Expression Vectors for Gene Function Analysis
(2001) BioTechniques Press, Westborough, Mass., 298 pp. (ISBN
1-881299-21-X). [0256] Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag, NY, 790 pp. (ISBN
3-540-41354-5). [0257] Old and Primrose, Principles of Gene
Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985)
Blackwell Scientific Publications, Boston, Mass. Studies in
Microbiology; V.2:409 pp. (ISBN 0-632-01318-4). [0258] Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.), Vols. 1-3 (ISBN
0-87969-309-6). [0259] Winnacker, From Genes To Clones:
Introduction To Gene Technology (1987) VCH Publishers, NY
(translated by Horst Ibelgaufts), 634 pp. (ISBN 0-89573-614-4).
INCORPORATION BY REFERENCE
[0260] The contents of all cited references (including literature
references, patents, patent applications, and websites) that maybe
cited throughout this application are hereby expressly incorporated
by reference in their entirety for any purpose, as are the
references cited therein. The practice of the present invention
will employ, unless otherwise indicated, conventional techniques of
immunology, molecular biology and cell biology, which are well
known in the art.
EQUIVALENTS
[0261] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 1
1
461153PRTHomo sapiens 1Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg
Asp Ser Gln Gln Lys 1 5 10 15 Ser Leu Val Met Ser Gly Pro Tyr Glu
Leu Lys Ala Leu His Leu Gln 20 25 30 Gly Gln Asp Met Glu Gln Gln
Val Val Phe Ser Met Ser Phe Val Gln 35 40 45 Gly Glu Glu Ser Asn
Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu 50 55 60 Lys Asn Leu
Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 65 70 75 80 Gln
Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu 85 90
95 Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe
100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln
Ala Glu 115 120 125 Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly
Gln Asp Ile Thr 130 135 140 Asp Phe Thr Met Gln Phe Val Ser Ser 145
150 2330PRTHomo sapiens 2Ala Ser Thr Lys Gly Pro Ser Val Phe Phe
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 3330PRTHomo sapiens
3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 4106PRTHomo sapiens 4Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55
60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
5105PRTHomo sapiens 5Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90
95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 64PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Phe
Gly Xaa Gly 1 79PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
85PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Leu Glu Trp Ile Gly 1 5 94PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Trp
Gly Xaa Gly 1 1030PRTHomo sapiens 10Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 1114PRTHomo sapiens 11Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10
1232PRTHomo sapiens 12Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala Arg 20 25 30 1311PRTHomo sapiens 13Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 1430PRTHomo sapiens
14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser 20
25 30 1514PRTHomo sapiens 15Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser 1 5 10 1632PRTHomo sapiens 16Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
1711PRTHomo sapiens 17Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 1823PRTHomo sapiens 18Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1915PRTHomo sapiens 19Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 1 5 10 15 2032PRTHomo sapiens 20Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 20 25 30
2110PRTHomo sapiens 21Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5
10 2223PRTHomo sapiens 22Asp Val Val Met Thr Gln Ser Pro Ala Phe
Leu Ser Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys 20
2315PRTHomo sapiens 23Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys
Leu Leu Ile Lys 1 5 10 15 2432PRTHomo sapiens 24Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile
Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30
2510PRTHomo sapiens 25Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5
10 26122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Gln Val His Leu Lys Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly
Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Lys Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120
27107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Glu Thr Thr Val Thr Gln Ser Pro Ala Ser Leu
Ser Met Ala Ile Gly 1 5 10 15 Glu Lys Val Thr Ile Arg Cys Ile Thr
Ser Thr Asp Ile Asp Val Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys
Pro Gly Glu Pro Pro Lys Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr
Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Val Phe Ile Ile Glu Asn Met Leu Ser 65 70 75 80 Glu
Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90
95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105
28122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Gly
Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
29122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Gly
Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
30122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Phe Ser Leu Ser Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly
Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Leu
Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Lys Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
31122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Val Ser Asp Tyr 20 25 30 Gly Val Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Trp Gly
Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
32122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30 Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Leu Ile Trp Gly Gly Gly Asp Thr Tyr Tyr Asn
Ser Pro Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Arg Thr Leu Trp
Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 33122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Asp
Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly Gly Gly Asp Thr Tyr Tyr
Asn Ser Pro Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn
Ser Lys Ser Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Arg Thr Leu
Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 34107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 35107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 36107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
36Asp Thr Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys
Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 37107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
37Glu Thr Thr Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys
Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 38107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
38Asp Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1
5 10 15 Glu Lys Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys
Leu Leu Ile 35 40 45 Lys Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 39107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Asp Thr Val Val Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1
5 10 15 Glu Lys Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Asp Gln Pro Pro Lys
Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 40107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Glu Thr Thr Val Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1
5 10 15 Glu Lys Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Val
Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Asp Gln Pro Pro Lys
Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro
Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Asp
Tyr Gly Val Ser 1 5 4216PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Leu Ile Trp Gly Gly Gly Asp
Thr Tyr Tyr Asn Ser Pro Leu Lys Ser 1 5 10 15 4314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Gln
Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr 1 5 10
4411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Ile Thr Ser Thr Asp Ile Asp Val Asp Met Asn 1 5
10 457PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Gln Gly Asn Thr Leu Arg Pro 1 5
469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Leu Gln Ser Asp Asn Leu Pro Leu Thr 1 5
* * * * *
References