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USE OF HMGB FRAGMENTS AS ANTI-INFLAMMATORY AGENTS
RELATED APPLICATIONS .
This application claims the benefit of U.S. Provisional Application Nos.
60/427,841 and 60!427,846, both of which were filed on November 20, 2002. The
entire teachings of both applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Inflammation is often induced by proinflammatory cytokines, such as tumor
necrosis factor (TNF), interleukin (IL)-la, IL-1(3, IL-6, platelet-activating
factor
(PAF), macrophage migration inhibitory factor (MIF), and other compounds.
These
proinflarmnatory cytolcines are produced by several different cell types, most
importantly immune cells (for example, monocytes, macrophages and
neutrophils),
but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle
cells,
epithelial cells, and neurons. These proinflammatory cytokines contribute to
various
disorders during the early stages of an inflammatory cytolcine cascade.
Inflammatory cytokine cascades contribute to deleterious characteristics,
including inflammation and apoptosis, of numerous disorders. Included are
disorders characterized by both localized and systemic reactions, including,
without
limitation, diseases involving the gastrointestinal tract and associated
tissues (such
as appendicitis, peptic, gastric and duodenal ulcers, peritonitis,
pancreatitis,
ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis,
epiglottitis,
achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's
disease,
enteritis, and Whipple's disease); systemic or local inflammatory diseases and
conditions (such as asthma, allergy, anaphylactic shoclc, immune complex
disease,
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organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis,
septicemia,
endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma,
granulomatosis,
and sarcoidosis); diseases involving the urogenital system and associated
tissues
(such as septic abortion, epididymitis, vaginitis, prostatitis, and
urethritis); diseases
involving the respiratory system and associated tissues (such as bronchitis,
emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress
syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis,
bronchiolitis,
pharyngitis, pleurisy, and sinusitis); diseases arising from infection by
various
viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B
virus,
hepatitis C virus and herpes), bacteria (such as disseminated bacteremia,
Dengue
fever), fungi (such as candidiasis) and protozoal and multicellular parasites
(such as
malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases
and
conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn,
urticaria
warts, and wheals); diseases involving the cardiovascular system and
associated
tissues (such as vasulitis, angiitis, endocarditis, arteritis,
atherosclerosis, restenosis,
thrombophlebitis, pericarditis, congestive heart failure, myocarditis,
myocardial
ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the
central or
peripheral nervous system and associated tissues (such as Alzheimer's disease,
meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral
embolism,
Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis,
and
uveitis); diseases of the bones, joints, muscles and connective tissues (such
as the
various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's
disease, gout,
periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune
and
inflammatory disorders (such as myasthenia gravis, tlnyoiditis, systemic lupus
erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft
rejection,
graft-versus-host disease, Type I diabetes, anleylosing spondylitis, Berger's
disease,
and Retier's syndrome); as well as various cancers, tumors and proliferative
disorders (such as Hodgkins disease); and, in any case the inflammatory or
immune
host response to any primary disease.
The early proinflammatory cytolcines (e.g., TNF, IL-1, etc.) mediate
inflammation, and induce the late release of high mobility group box 1 (HMGB1)
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(also known as HMG-1 and HMG1), a protein that accumulates in serum and
mediates delayed lethality and further induction of early proinflammatory
cytokines.
HMGB 1 was first identified as the founding member of a family of DNA-
binding proteins termed lugh mobility group box (HMGB) proteins that are
critical
for DNA structure and stability. It was identified nearly 40 years ago as a
ubiquitously expressed nuclear protein that binds double-stranded DNA without
sequence specificity.
HMGB 1 binding bends DNA to promote formation and stability of
nucleoprotein complexes that facilitate gene transcription of glucocorticoid
receptors
and RAG recombinase. The HMGB 1 molecule has three domains: two DNA
binding motifs termed HMGB A and HMGB B boxes, and an acidic carboxyl
terminus. The two HMGB boxes are highly conserved ~0 amino acid, L-shaped
domains. HMGB boxes are also expressed in other transcription factors
including
the RNA polymerase I transcription factor human upstream-binding factor and
lymphoid-specific factor.
Recent evidence has implicated HMGB 1 as a cytolcine mediator of
inflammatory conditions. For example, HMGB 1 has been implicated as a cytokine
mediator of delayed lethality in endotoxemia. That work demonstrated that
bacterial
endotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages to
release
HMGB 1 as a late response to activation, resulting in elevated serum HMGB 1
levels
that are toxic. Antibodies against HMGB 1 prevent' lethality of endotoxin even
when
antibody administration is delayed until after the early cytokine response.
Like other
proinflammatory cytokines, HMGB 1 is a potent activator of monocytes.
Intratracheal application of HMGB 1 causes acute lung injury, and anti-HMGB 1
antibodies protect against endotoxin-induced lung edema. Serum HMGB 1 levels
are
elevated in critically ill patients with sepsis or hemorrhagic shoclc, and
levels are
significantly higher in non-survivors as compared to survivors.
HMGB 1 has also been implicated as a ligand for RAGE, a multi-ligand
receptor of the immunoglobulin superfamily. RAGE is expressed on endothelial
cells, smooth muscle cells, monocytes, and nerves, and ligand interaction
transduces
signals through MAP kinase, P21 ras, and NF-xB. The delayed kinetics of HMGB 1
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appearance during endotoxemia makes it a potentially good therapeutic target,
but
little is known about the molecular basis of HMGB 1 signaling and toxicity.
Therefore, it would be useful to identify characteristics of HMGB 1
proinflammatory activity, particularly the active domains) responsible for
this
activity, and airy inhibitory effects of other domains.
SUMMARY OF THE INVENTION
The present invention is based on the discoveries that (1) the HMGB A box
serves as a competitive inhibitor of HMGB proinflammatory action, (2) the HMGB
B box has the predominant proinflammatory activity of HMGB, and (3)
combination
therapies involving agents that inhibit HMGB biological activity and agents
that
inhibit TNF biological activity can be used for the treatment of conditions
characterized by activation of the inflammatory cytolcine cascade. Agents that
inhibit HMGB biological activity include the HMGB A box, which serves as a
competitive inhibitor of HMGB prointlammatory action, and antibodies to HMGB,
for example, the HMGB B box.
Accordingly, in one embodiment, the invention is a polypeptide comprising a
high mobility group box protein (HMGB) A box or variant thereof, or an A box
biologically active fragment or variant thereof, which can inhibit release of
a
proinflammatory cytolcine from a cell treated with high mobility group box
(HMGB)
protein, wherein the HMGB A box is selected from the group consisting of an
HMG1L5 (formerly HMG1L10) A box, an HMG1L1 A box, an HMG1L4 A box, an
HMGB A box polypeptide of BAC clone RPl 1-395A23, an HMG1L9 A box, an
LOC122441 A box, an LOC139603 A box, and an HMG1L~ A box. In one
embodiment, the polypeptide can be in a pharmaceutically acceptable carrier.
In another embodiment, the invention is a purified preparation of antibodies
that specifically bind to a high mobility group box protein (HMGB) B box but
do not
specifically bind to non-B box epitopes of HMGB, wherein the antibodies can
inhibit release of a proinflammatory cytokine from a cell treated with HMGB,
wherein the HMGB B box is selected from the group consisting of an HMG1L5
(formerly HMG1L10) B box, an HMG1L1 B box, an HMG1L4 B box, and an
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HMGB B box polypeptide of BAC clone RP11-395A23. In one embodiment, the
antibodies can be in a pharmaceutically acceptable carrier.
In still another embodiment, the invention is a polypeptide comprising a high
mobility group box protein (HMGB) B box or variant thereof, or a B box
biologically active fragment or variant thereof, but not comprising a full
length
HMGB, wherein the polypeptide can cause release of a proinflammatory cytokine
from a cell, and wherein the HMGB B box is selected from the group consisting
of
an HMG1L5 (formerly HMG1L10) B box, an HMG1L1 B box, an HMG1L4 B box,
and an HMGB B box polypeptide of BAC clone RP11-395A23. In one
embodiment, the polypeptide can be in a pharmaceutically acceptable carrier.
In other embodiments, the invention comprises vectors encoding the
polypeptides described above.
In still another embodiment, the invention is a method of inhibiting release
of
a proinflaxnmatory cytolcine from a mammalian cell, the method comprising
treating
the cell with an amount of a purified preparation of antibodies that
specifically bind
to a high mobility group box protein (HMGB) B box but do not specifically bind
to
non-B box epitopes of HMGB, wherein the HMGB B box is selected from the group
consisting of an HMG1L5 (formerly HMG1L10) B box, an HMG1L1 B box, an
HMG1L4 B box, and an HMGB B box polypeptide of BAC clone RP11-395A23.
In another embodiment, the invention is a method of inhibiting release of a
proinflammatory cytolcine from a mammalian cell, the method comprising
treating
the cell with a polypeptide comprising a high mobility group box protein
(HMGB) A
box or variant thereof, or an A box biologically active fragment or variant
thereof,
which can inhibit release of a proinflammatory cytolcine from a cell treated
with high
mobility group box (HMGB) protein in an amount sufficient to inhibit release
of the
proinflammatory cytokine from the cell, wherein the HMGB A box is selected
from
the group consisting of an HMG1L5 (formerly HMG1L10) A box, an HMG1L1 A
box, an HMG1L4 A box, an HMGB A box polypeptide of BAC clone RP11-
395A23, an HMG1L9 A box, an LOC122441 A box, an LOC139603 A box, and an
HMG1L8 A box. In one embodiment, the cell can be treated with a vector
encoding
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a polypeptide comprising the A box polypeptide, A box biologically active
fragment,
or variant thereof.
In another embodiment, the invention is a method of treating a condition in a
patient characterized by activation of an inflammatory cytokine cascade,
comprising
administering to the patient a purified preparation of antibodies that
specifically bind
to a high mobility group box protein (HMGB) B box but do not specifically bind
to
non-B box epitopes of HMGB, in an amount sufficient to inhibit the
inflammatory
cytolcine cascade, wherein the HMGB B box is selected from the group
consisting of
an HMG1L5 (formerly HMG1L10) B box, an HMG1L1 B box, an HMG1L4 B box,
and an HMGB B box polypeptide of BAC clone RP11-395A23.
In another embodiment, the invention is a method of treating a condition in a
patient characterized by activation of an inflammatory cytokine cascade,
comprising
administering to the patient a polypeptide comprising a high mobility group
box
protein (HMGB) A box or variant thereof, or an A box biologically active
fragment
or variant thereof, which can inhibit release of a proinflammatory cytolcine
from a
cell treated with high mobility group box (HMGB) protein, in an amount
sufficient
to inhibit release of the proinflammatory cytokine from the cell, wherein the
HMGB
A box is selected from the group consisting of an HMG1L5 (formerly HMG1L10) A
box, an HMG1L1 A box, an HMG1L4 A box, an HMGB A box polypeptide of BAC
clone RP11-395A23, an HMG1L9 A box, an LOC122441 B box, an LOC139603 A
box, and an HMG1L8 A box.
In still another embodiment, the invention is a method of stimulating the
release of a proinflammatory cytokine from a cell comprising treating the cell
with a
polypeptide comprising a high mobility group box protein (HMGB) B box or
variant
thereof, or a B box biologically active fragment thereof, but not comprising a
full
length HMGB, in an amount sufficient to stimulate the release of the
proinflammatory cytolcine from the cell, wherein the HMGB B box is selected
from
the group consisting of an HMG1L5 (formerly HMG1L10) B box, an HMG1L1 B
box, an HMG1L4 B box, and an HMGB B box polypeptide of BAC clone RP11-
395A23. In one embodiment, the cell can be treated with a vector encoding a
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polypeptide comprising the B box polypeptide, B box biologically active
fragment,
or variant thereof.
In still another embodiment, the invention is a method for effecting weight
loss or treating obesity in a patient, comprising administering to the patient
an
effective amount of a polypeptide comprising a high mobility group box protein
(HMGB) B box or variant thereof, or a B box biologically active fragment or
variant
thereof, but not comprising a full length HMGB polypeptide, in an amount
sufficient to stimulate the release of a proinflammatory cytokine from a cell,
wherein the HMGB B box is selected from the group consisting of an HMG1L5
(formerly HMG 1 L 10) B box, an HMGl L 1 B box, an HMG 1 L4 B box, and an
HMGB B box polypeptide of BAC clone RP11-395A23.
In another embodiment, the invention is a method of determining whether a
compound inhibits inflammation, comprising combining the compound with a) a
cell that releases a proinflammatory cytokine when exposed to a high mobility
group
box protein (HMGB) B box or a biologically active fragment thereof; and b) the
HMGB B box or biologically active fragment thereof, wherein said HMGB B box is
selected from the group consisting of an HMG1L5 (formerly HMG1L10) B box, an
HMG1L1 B box, an HMG1L4 B box, and an HMGB B box polypeptide of BAC
clone RP11-395A23; then determining whether the compound inhibits the release
of
the proinflammatory cytokine from the cell.
In yet another embodiment, the invention is a pharmaceutical composition
comprising a polypeptide comprising a high mobility group box (HMGB) A box, or
a fragment or variant thereof, that can inhibit release of a proinflammatory
cytokine
from a cell treated with a high mobility group box (HMGB) protein and an agent
that inhibits TNF biological activity, where the agent is selected from the
group
consisting of infliximab, etanercept, adalimumab, CDP~70, CDP571, Lenercept,
and Thalidomide, in a pharmaceutically acceptable carrier. The HMGB A box is
preferably a vertebrate HMGB A box, for example, a mammalian HMGB A box,
more preferably, a mammalian HMGB 1 A box, for example, a human HMGB 1 A
box, and most preferably, the HMGB1 A box comprising or consisting of the
sequence of SEQ ID N0:4, SEQ ID N0:22, or SEQ ID N0:57.
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In another embodiment, the invention is a pharmaceutical composition
comprising an antibody that binds an HMGB polypeptide or a biologically active
fragment thereof, for example, an HMGB B box polypeptide or biologically
active
fragment thereof, and an agent that inhibits TNF biological activity, where
the agent
is selected from the group consisting of infliximab, etanercept, adalimumab,
CDP870, CDP571, Lenercept, and Thalidomide, in a pharmaceutically acceptable
carrier.
In still another embodiment, the invention is a method of treating a condition
in a patient characterized by activation of an inflammatory cytokine cascade
comprising administering to the patient a composition comprising a polypeptide
comprising a high mobility group box (HMGB) A box or a fragment or variant
thereof that can inhibit release of a proinflammatory cytokine from a cell
treated
with high mobility group box (HMGB) protein and an agent that inhibits TNF
biological activity, where the agent is selected from the group consisting of
infliximab, etanercept, adalimumab, CDP870, CDP571, Lenercept, and
Thalidomide.
In still another embodiment, the invention is a method of treating a condition
in a patient characterized by activation of an inflammatory cytokine cascade
comprising administering to the patient a composition comprising an antibody
that
binds an HMGB polypeptide or a biologically active fragment thereof, for
example,
an HMGB B box polypeptide or a biologically active fragment thereof, and an
agent
that inhibits TNF biological activity, where the agent is selected from the
group
consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, Lenercept,
and Thalidomide.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of HMGB 1 mutants and their activity in
TNF release (pg/ml).
FIG. 2A is a histogram showing the effect of 0 ~glml, 0.01 ~,glml, 0.1 ~,glml,
1 ~,g/ml or 10 ~,g/ml of B box on TNF release (pg/ml) in RAW 264.7 cells.
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FIG. 2B is a histogram showing the effect of 0 ~g/ml, 0.01 ~,g/ml, 0.1 wg/ml,
1 ~,g/ml or 10 ~.g/ml of B box on IL-1(3 release (pg/ml) in RAW 264.7 cells.
FIG. 2C is a histogram showing the effect of 0 wg/ml, 0.01 wg/ml, 0.1
~,g/ml, 1 ~,g/ml or 10 ~g/ml of B box on IL-6 release (pg/nil) in RAW 264.7
cells.
FIG. 2D a scanned image of a blot of an RNAse protection assay, showing
the effect of B box (at 0 hours, 4 hours, 8 hours, or 24 hours after
administration) or
vector alone (at 4 hours after administration) on TNF rnRNA expression in RAW
264.7 cells.
FIG. 2E is a histogram of the effect of HMGB 1 B box on TNF protein
release (pg/ml) from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24 hours,
32
hours or 48 hours after administration.
FIG. 2F is a histogram of the effect of vector on TNF protein release (pg/ml)
from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24 hours, 32 hours or 48
hours
after administration.
FIG. 3 is a schematic representation of HMGB 1 B box mutants and their
activity in T'NF release (pg/ml).
FIG. 4A is a graph of the effect of 0 ~,g/ml, 5 wg/ml, 10 ~.g/ml, or 25 ~,g/ml
of HMG1 A box protein on the release of TNF (as a percent of HMGB 1 mediated
TNF release alone) from RAW 264.7 cells.
FIG. 4B is a histogram of the effect of HMGB 1 (0 or 1.5 ~g/ml), HMGB 1 A
box (0 or 10 ~,g/ml), or vector (0 or 10 ~,g/ml), alone, or in combination, on
the
release of TNF (as a percent of HMGB 1 mediated TNF release alone) from RAW
264.7 cells.
FIG. SA is a graph of binding of'zsI_HMGB 1 binding to RAW 264.7 cells
(CPM/well) over time (minutes).
FIG. SB is a histogram of the binding of lzsl_HMGB1 in the absence of
unlabeled HMGB 1 or HMGB 1 A box for 2 hours at 4°C (Total), or in the
presence
of 5,000 molar excess of unlabeled HMGB1 (HMGB1) or A box (A box), measured
as a percent of the total CPM/well.
FIG. 6 is a histogram of the effects of HMGB 1 (HMG-1; 0 ~.glml or 1
~,g/ml) or HMGB 1 B box (B Box; 0 ~glml or 10 ~,g/ml), alone or in combination
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with anti-B box antibody (25 ~.g/ml or 100 ~g/ml) or IgG (25 ~.glml or 100
~g/ml)
on TNF release from R.AW 264.7 cells (expressed as a percent of HMGB 1
mediated
TNF release alone).
FIG. 7A is a scanned image of a hematoxylin and eosin stained kidney
section obtained from an untreated mouse.
FIG. 7B is a scanned image of a hematoxylin and eosin stained kidney
section obtained from a mouse administered HMGB 1 B box.
FIG. 7C is a scanned image of a hematoxylin and eosin stained myocardium
section obtained from an untreated mouse.
FIG. 7D is a scanned image of a hematoxylin and eosin stained myocardium
section obtained from a mouse administered HMGB 1 B box.
FIG. 7E is a scanned image of a hematoxylin and eosin stained lung section
obtained from an untreated mouse.
FIG. 7F is a scanned image of a hematoxylin and eosin stained lung section
obtained from a mouse administered HMGB 1 B box.
FIG. 7G is a scanned image of a hematoxylin and eosin stained liver section
obtained from an untreated mouse.
FIG. 7H is a scanned image of a hematoxylin and eosin stained liver section
obtained from a mouse administered HMGB 1 B box.
FIG. 7I is a scanned image of a hematoxylin and eosin stained liver section
(high magnification) obtained from an untreated mouse.
FIG. 7J is a scanned image of a hematoxylin and eosin stained liver section
(high magnification) obtained from a mouse administered HMGB 1 B box.
FIG. 8 is a graph of the level of HMGB 1 (ng/ml) in mice subjected to cecal
ligation and puncture (CLP) over time (hours).
FIG. 9 is a graph of the effect of HMGB A Box (60 wg/mouse or 600
~.g/mouse) or no treatment on survival of mice over time (days) after cecal
ligation
and puncture (CLP).
FIG. 1 OA is a graph of the effect of anti-HMGB 1 antibody (dark circles) or
no treatment (open circles) on survival of mice over time (days) after cecal
ligation
and puncture (CLP).
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FIG. lOB is a graph of the effect of anti-HMGB1 B box antiserum (~) or no
treatment (*) on the survival (days) of mice administered lipopolysaccharide
(LPS).
FIG. 1 lA is a histogram of the effect of anti-RAGE antibody or non-immune
IgG on TNF release from RAW 264.7 cells treated with HMGB1 (HMG-1),
lipopolysacchaxide (LPS), or HMGB1 B box (B box).
FIG. 11B is a histogram of the effect of HMGBl (HMG-1) or HMGB1 B
box (B Box) polypeptide stimulation on activation of the NF-xB-dependent ELAM
promoter (measured by luciferase activity) in RAW 264.7 cells co-transfected
with a
marine MyD 88-dominant negative (+MyD 88 DN) mutant (corresponding to amino
acids 146-296), or empty vector (-MyD 88 DN). Data axe expressed as the ratio
(fold-activation) of average luciferase values from unstimulated and
stimulated cells
(subtracted for background) + SD.
FIG. 12A is the amino acid sequence of a human HMG1 polypeptide (SEQ
ID NO:1).
FIG. 12B is the amino acid sequence of rat and mouse HMG1 (SEQ ID
N0:2).
FIG. 12C is the amino acid sequence of human HMG2 (SEQ ID NO:3).
FIG. 12D is the amino acid sequence of a human, mouse, and rat HMG1 A
box polypeptide (SEQ ID N0:4).
FIG. 12E is the amino acid sequence of a human, mouse, and rat HMG1 B
box polypeptide (SEQ ID NO:S).
FIG. 12F is the nucleic acid sequence of a forward primer for human HMGl
(SEQ ID N0:6).
FIG. 12G is the nucleic acid sequence of a reverse primer for human HMG1
(SEQ ID N0:7).
FIG. 12H is the nucleic acid sequence of a forward primer for the carboxy
terminus mutant of human HMGl (SEQ ID N0:8).
FIG. 12I is the nucleic acid sequence of a reverse primer for the carboxy
terminus mutant of human HMG1 (SEQ ID N0:9).
FIG. 12J is the nucleic acid sequence of a forward primer for the amino
terminus plus B box mutant of human HMGl (SEQ ID NO:10).
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FIG. 12K is the nucleic acid sequence of a reverse primer for the amino
terminus plus B box mutant of human HMG1 (SEQ ID NO:11).
FIG. 12L is the nucleic acid sequence of a forward primer for a B box
mutant of human HMG1 (SEQ ID N0:12).
FIG. 12M is the nucleic acid sequence of a reverse primer for a B box
mutant of human HMGl (SEQ ID N0:13).
FIG. 12N is the nucleic acid sequence of a forward primer for the amino
terminus plus A box mutant of human HMGl (SEQ ID N0:14).
FIG. 120 is the nucleic acid sequence of a reverse primer for the amino
terminus plus A box mutant of human HMG1 (SEQ ID NO:15).
FIG. 13 is a sequence alignment of HMGB 1 polypeptide sequences from rat
(SEQ ID N0:2), mouse (SEQ ID NO:2), and human (SEQ ID N0:18).
FIG. 14A is the nucleic acid sequence of HMG1L5 (formerly HMG1L10)
(SEQ ID NO: 32) encoding an HMGB polypeptide.
FIG. 14B is the polypeptide sequence of HMG1L5 (formerly HMG1L10)
(SEQ ID NO: 24) encoding an HMGB polypeptide.
FIG. 14C is the nucleic acid sequence of HMG1L1 (SEQ ID NO: 33)
encoding an HMGB polypeptide.
FIG. 14D is the polypeptide sequence of HMG1L1 (SEQ ID NO: 25)
encoding an HMGB polypeptide.
FIG. 14E is the nucleic acid sequence of HMG1L4 (SEQ ID NO: 34)
encoding an HMGB polypeptide.
FIG. 14F is the polypeptide sequence of HMG1L4 (SEQ ID NO: 26)
encoding an HMGB polypeptide.
FIG. 14G is the nucleic acid sequence of the HMG polypeptide sequence of
the BAC clone RP11-395A23 (SEQ ID NO: 35).
FIG. 14H is the polypeptide sequence of the HMG polypeptide sequence of
the BAC clone RP11-395A23 (SEQ ID NO: 27) encoding an HMGB polypeptide.
FIG. 14I is the nucleic acid sequence of HMG1L9 (SEQ ID NO: 36)
encoding an HMGB polypeptide.
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FIG. 14J is the polypeptide sequence of HMG1L9 (SEQ ID NO: 28)
encoding an HMGB polypeptide.
FIG. 14I~ is the nucleic acid sequence of LOC122441 (SEQ ID NO: 37)
encoding an HMGB polypeptide.
FIG. 14L is the polypeptide sequence of LOC122441 (SEQ ID NO: 29)
encoding an HMGB polypeptide.
FIG. 14M is the nucleic acid sequence of LOC139603 (SEQ ID NO: 38)
encoding an HMGB polypeptide.
FIG. 14N is the polypeptide sequence of LOC139603 (SEQ ID NO: 30)
encoding an HMGB polypeptide.
FIG. 140 is the nucleic acid sequence of HMG1L8 (SEQ ID NO: 39)
encoding an HMGB polypeptide.
FIG. 14P is the polypeptide sequence of HMG1L8 (SEQ ID NO: 31)
encoding an HMGB polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of cell culture, molecular biology,
microbiology,
cell biology, and immunology, which are well within the skill of the art. Such
techniques are fully explained in the literature. See, e.g., Sambrook et al.,
1989,
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press;
Ausubel et al. (1995), "Short Protocols in Molecular Biology", John Wiley and
Sons; Methods in Enzymology (several volumes); Methods in Cell Biology
(several
volumes), and Methods in Molecular Biology (several volumes).
The present invention is based on a series of discoveries that further
elucidate various characteristics of the ability of HMGB 1 to induce
production of
proinflammatory cytokines and inflammatory cytolcine cascades. Specifically,
it has
been discovered that the proinflammatory active domain of HMGB 1 is the B box
(and in particular, the first 20 amino acids of the B box), and that
antibodies specific
to the B box will inhibit proinflammatory cytolcine release and inflammatory
cytolcine cascades, with results that can alleviate deleterious symptoms
caused by
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inflammatory cytolcine cascades. It has also been discovered that the A box is
a
weak agonist of inflammatory cytokine release, and competitively inhibits the
proinflammatory activity of the B box and of HMGB 1. It has further been
discovered that inhibitors of TNF biological activity can be combined with
HMGB
A boxes and/or antibodies to HMGB1, to form pharmaceutical compositions for
use
in treating conditions characterized by activation of an inflammatory cytokine
cascade in patients.
As used herein, an "HMGB polypeptide" or an "HMGB protein" is a
substantially pure, or substantially pure and isolated polypeptide, that has
been
separated from components that naturally accompany it, or a synthetically or
recombinantly produced polypeptide having the same amino acid sequence, and
increases inflammation, and/or increases release of a proinflammatory cytokine
from a cell, and/or increases the activity of the inflammatory cytokine
cascade. In
one embodiment, the HMGB polypeptide has one of the above biological
activities.
In another embodiment, the HMGB polypeptide has two of the above biological
activities. In a third embodiment, the HMGB polypeptide has all three of the
above
biological activities.
Preferably, the HMGB polypeptide is a mammalian HMGB polypeptide, for
example, a human HMGB 1 polypeptide. Examples of an HMGB polypeptide
include a polypeptide comprising or consisting of the sequence of SEQ ID NO:1,
SEQ ID N0:2, SEQ ID N0:3, or SEQ ID N0:18. Preferably, the HMGB
polypeptide contains a B box DNA binding domain and/or an A box DNA binding
domain, and/or an acidic carboxyl terminus as described herein. Other examples
of
HMGB polypeptides are described in GenBank Accession Numbers AAA64970,
AAB08987, P07155, AAA20508, 529857, P09429, NP 002119, CAA31110,
502826, U00431, X67668, NP 005333, NM 016957, and J04179, the entire
teachings of which are incorporated herein by reference. Additional examples
of
HMGB polypeptides include, but are not limited to mammalian HMG1 ((HMGB1)
as described, for example, in GenBank Accession Number U51677), HMG2
((HMGB2) as described, for example, in GenBanlc Accession Number M83665),
HMG-2A ((HMGB3, HMG-4) as described, for example, in GenBank Accession
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Numbers NM 005342 and NP 005333), HMG14 (as described, for example, in
GenBank Accession Number P05114), HMG17 (as described, for example, in
GenBank Accession Number X13546), HMGI (as described, for example, in
GenBanlc Accession Number L17131), and HMGY (as described, for example, in
GenBank Accession Number M23618); nonmammalian HMG T1 (as described, for
example, in GenBank Accession Number X02666) and HMG T2 (as described, for
example, in GenBank Accession Number L32859) (rainbow trout); HMG-X (as
described, for example, in GenBanlc Accession Number D30765) (Xenopus), HMG
D (as described, for example, in GenBank Accession Number X71138) and HMG Z
(as described, for example, in GenBank Accession Number X71139) (Drosophila);
NHP10 protein (HMG protein homolog NHP 1) (as described, for example, in
GenBank Accession Number 248008) (yeast); non-histone chromosomal protein (as
described, for example, in GenBank Accession Number 000479) (yeast); HMG 1/ 2
like protein (as described, for example, in GenBank Accession Number 211540)
(wheat, maize, soybean); upstream binding factor (UBF-1) (as described, for
example, in GenBank Accession Number X53390); PMS1 protein homolog 1 (as
described, for example, in GenBank Accession Number U13695); single-strand
recognition protein (SSRP, structure-specific recognition protein) (as
described, for
example, in GenBanlc Accession Number M86737); the HMG homolog TDP-1 (as
described, for example, in GenBanlc Accession Number M74017); mammalian
sex-determining region Y protein (SRY, testis-determining factor) (as
described, for
example, in GenBanlc Accession Number X53772); fungal proteins: mat-1 (as
described, for example, in GenBanlc Accession Number AB009451), ste 11 (as
described, for example, in GenBank Accession Number X53431) and Mc 1; SOX 14
(as described, for example, in GenBanlc Accession Number AF107043), as well as
SOX 1 (as described, for example, in GenBank Accession Number Y13436), SOX 2
(as described, for example, in GenBanlc Accession Number 231560), SOX 3 (as
described, for example, in GenBanlc Accession Number X71135), SOX 6 (as
described, for example, in GenBanlc Accession Number AF309034), SOX 8 (as
described, for example, in GenBanlc Accession Number AF226675), SOX 10 (as
described, for example, in GenBanlc Accession Number AJ001183), SOX 12 (as
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described, for example, in GenBank Accession Ntunber X73039) and SOX 21 (as
described, for example, in GenBank Accession Number AF 107044)); lymphoid
specific factor (LEF-1) (as described, for example, in GenBank Accession
Number
X58636); T-cell specific transcription factor (TCF-1) (as described, for
example, in
GenBanlc Accession Number X59869); MTT1 (as described, for example, in
GenBank Accession Number M62810); amd SP100-HMG nuclear autoantigen (as
described, for example, in GenBank Accession Number U36501).
Other examples of HMGB proteins are polypeptides encoded by HMGB
nucleic acid sequences having GenBank Accession Numbers NG 000897 (HMG1L5
(formerly HMG1L10)) (and in particular by nucleotides 150-797 of NG 000897, as
shown in FIGS. 14A and 14B); AF076674 (HMG1L1) (and in particular by
nucleotides 1-633 of AF076674, as shown in FIGS. 14C and 14D; AF076676
(HMG1L4) (and in particular by nucleotides 1-564 of AF076676, as shown in
FIGS.
14E and 14F); AC010149 (HMG sequence from BAC clone RPl 1-395A23) (and in
particular by nucleotides 75503-76117 of AC010149), as shown in FIGS. 14G and
14H); AF165168 (HMG1L9) (and in particular by nucleotides 729-968 of
AF165168, _as shown in FIGS. 14I and 14J); XM_063129 (LOC122441) (and in
particular by nucleotides 319-558 of XM 063129, as shown in FIGS. 14I~ and
14L);
XM -066789 (LOC139603) (and in particular by nucleotides 1-258 of XM_066789,
as shown in FIGS. 14M and 14N); and AF165167 (HMG1L8) (and in particular by
nucleotides 456-666 of AF165167, as shown in FIGS. 140 and 14P)
The HMGB polypeptides of the present invention also encompass sequence
variants. Variants include a substantially homologous polypeptide encoded by
the
same genetic locus in an organism, i.e., an allelic variant, as well as other
variants.
Variants also encompass polypeptides derived from other genetic loci in an
organism, but having substantial homology to a polypeptide encoded by an HMGB
nucleic acid molecule, and complements and portions thereof, or having
substantial
homology to a polypeptide encoded by a nucleic acid molecule comprising the
nucleotide sequence of an HMGB nucleic acid molecule. Examples of HMGB
nucleic acid molecules are known in the art and can be derived from HMGB
polypeptides as described herein. Variants also include polypeptides
substantially
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homologous or identical to these polypeptides but derived from another
organism,
i.e., an ortholog. Variants also include polypeptides that are substantially
homologous or identical to these polypeptides that are produced by chemical
synthesis. Variants also include polypeptides that are substantially
homologous or
identical to these polypeptides that are produced by recombinant methods.
Preferably, the HMGB polypeptide has at least 60%, more preferably, at least
70%,
75%, 80%, 85%, or 90%, and most preferably at least 95%, sequence identity to
a
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ
ID N0:3 and SEQ ID N0:18, as determined using the BLAST program and
parameters described herein and one of more of the biological activities of an
HMGB
polypeptide.
In other embodiments, the present invention is directed to an HMGB
polypeptide fragment that has HMGB biological activity. By an "HMGB
polypeptide fragment that has HMGB biological activity" or a "biologically
active
HMGB fragment" is meant a fragment of an HMGB polypeptide that has the
activity
of an HMGB polypeptide. An example of such an HMGB polypeptide fragment is
the HMGB B box, as described herein. Biologically active HMGB fragments can be
generated using standard molecular biology techniques and assaying the
function of
the fragment by determining if the fragment, when administered to a cell,
increases
release of a proinflammatory cytokine from the cell, compared to a suitable
control,
for example, using methods described herein.
As used herein, an "HMGB A box", also referred to herein as an "A box", is
a substantially pure, or substantially pure and isolated polypeptide, that has
been
separated from components that naturally accompany it, and consists of an
amino
acid sequence that is less than a full length HMGB polypeptide and which has
one or
more of the following biological activities: inhibiting inflammation, and/or
inhibiting
release of a proinflammatory cytokine from a cell, and/or decreasing the
activity of
the inflammatory cytokine cascade. In one embodiment, the HMGB A box
polypeptide has one of the above biological activities. In another embodiment,
the
HMGB A box polypeptide has two of the above biological activities. In a third
embodiment, the HMGB A box polypeptide has all three of the above biological
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activities. Preferably, the HMGB A box has no more than 10%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 80%, or 90%, of the biological activity of a full length
HMGB
polypeptide. In one embodiment, the HMGB A box amino acid consists of the
sequence of SEQ ID N0:4, SEQ ID N0:22, or SEQ ID N0:57, or the amino acid
sequence in the corresponding region of an HMGB protein in a mammal.
An HMGB A box is also a recombinantly produced polypeptide having the
same amino acid sequence as the A box sequences described above. Preferably,
the
HMGB A box is a mammalian HMGB A box, for example, a human HMG1 A box.
The HMGB A box polypeptides of the present invention preferably comprise or
consist of the sequence of SEQ ID N0:4, SEQ ID N0:22, or SEQ ID N0:57, or the
amino acid sequence in the corresponding region of an HMGB protein in a
mammal.
An HMGB A box often has no moxe than about 85 amino acids and no fewer than
about 4 amino acids. Examples of polypeptides having A box sequences within
them
include, but are not limited to, the HMGB proteins and polypeptides described
herein. The A box sequences in such polypeptides can be determined and
isolated
using methods described herein, for example, by sequence comparisons to A
boxes
described herein and testing for A box biological activity using methods
described
herein or other methods known in the art.
Additional examples of HMGB A box polypeptide sequences include the
following sequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human HMGB1; SEQ ID NO: 40);
DSSVNFAEF SKKCSERWKT MSAKEKSKFE DMAKSDKARY DREMKNYVPP
KGDK (human HMGB2; SEQ ID NO: 41); PEVPVNFAEF SKKCSERWKT
VSGKEKSKFD EMAKADKVRY DREMKDYGPA KGGK (human HMGB3; SEQ
ID NO: 42); PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY
EREMKTYIPP KGET (HMG1L5 (formerly HMG1L10); SEQ ID NO: 43);
SDASVNFSEF SNKCSERWKT MSAKEKGKFE DMAKADKTHY
ERQMKTYIPP KGET (HMG1L1; SEQ ID NO: 44); PDASVNFSEF
SKKCSERWKA MSAKDKGKFE DMAKVDKADY EREMKTYIPP KGET
(HMG1L4; SEQ ID NO: 45); PDASVKFSEF LKKCSETWKT IFAKEKGKFE
DMAKADKAHY EREMKTYIPP KGEK (HMG sequence from BAC clone RP11-
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395A23; SEQ ID NO: 46); PDASINFSEF SQKCPETWKT TIAKEKGKFE
DMAKADKAHY EREMKTYIPP KGET (HMG1L9; SEQ ID NO: 47);
PDASVNSSEF SKKCSERWKTMPTKQGKFE DMAKADRAH (HMG1L8; SEQ
ID NO: 48); PDASVNFSEF SKKCLVRGKT MSAKEKGQFE AMAR.ADKARY
EREMKTYIP PKGET (LOC122441; SEQ ID NO: 49); LDASVSFSEF
SNKCSERWKT MSVKEKGKFE DMAKADKACY EREMKIYPYL KGRQ
(LOC139603; SEQ ID NO: 50); and GKGDPKKPRG KMSSYAFFVQ
TCREEHKKKH PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human HMGB 1 A box; SEQ ID NO: 57).
The HMGB A box polypeptides of the present invention also encompass
sequence variants. Variants include a substantially homologous polypeptide
encoded
by the same genetic locus in an organism, i.e., an allelic variant, as well as
other
variants. Variants also encompass polypeptides derived from other genetic loci
in an
organism, but having substantial homology to a polypeptide encoded by an HMGB
A
box nucleic acid molecule, and complements and portions thereof, or having
substantial homology to a polypeptide encoded by a nucleic acid molecule
comprising the nucleotide sequence of an HMGB A box nucleic acid molecule.
Examples of HMGB A box nucleic acid molecules are lcnown in the art and can be
derived from HMGB A polypeptides as described herein. Variants also include
polypeptides substantially homologous or identical to these polypeptides but
derived
from another organism, i.e., an ortholog. Variants also include polypeptides
that are
substantially homologous or identical to these polypeptides that are produced
by
chemical synthesis. Variants also include polypeptides that are substantially
homologous or identical to these polypeptides that are produced by recombinant
methods. Preferably, an HMGB A box has at least 60%, more preferably, at least
70%, 75%, 80%, 85%, or 90%, and most preferably at least 95%, sequence
identity
to an HMGB A box polypeptide described herein, for example, the sequence of
SEQ
ID N0:4, SEQ ID N0:22, or SEQ ID N0:57, as determined using the BLAST
program and parameters described herein, and one of more of the biological
activities
of an HMGB A box, as determined using methods described herein or other method
known in the art.
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The present invention also features A box biologically active fragments. By
an "A box fragment that has A box biological activity" or an "A box
biologically
active fragment" is meant a fragment of an HMGB A box that has the activity of
an
HMGB A box, as described herein. For example, the A box fragment can decrease
release of a pro-inflammatory cytokine from a vertebrate cell, decrease
inflammation,
and/or decrease activity of the inflammatory cytokine cascade. A box fragments
can
be generated using standard molecular biology techniques and assaying the
function
of the fragment by determining if the fragment, when administered to a cell
inhibits
release of a proinflammatory cytokine from the cell, for example, using
methods
described herein. A box biologically active fragments can be used in the
methods
described herein in which full length A box polypeptides are used, for
example,
inhibiting release of a proinflammatory cytokine from a cell, or treating a
patient
having a condition characterized by activation of an inflammatory cytokine
cascade.
As used herein, an "HMGB B box", also referred to herein as a "B box", is a
substantially pure, or substantially pure and isolated polypeptide, that has
been
separated from components that naturally accompany it, and consists of an
amino
acid sequence that is less than a full length HMGB polypeptide and has one or
more
of the following biological activities: increasing inflammation, increasing
release of a
proinflammatory cytolcine from a cell, and or increasing the activity of the
inflammatory cytokine cascade. In one embodiment, the HMGB B box polypeptide
has one of the above biological activities. In another embodiment, the HMGB B
box
polypeptide has two of the above biological activities. In a third embodiment,
the
HMGB B box polypeptide has all three of the above biological activities.
Preferably,
the HMGB B box has at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, of the
biological activity of a full length HMGB polypeptide. In another embodiment,
the
HMGB B box does not comprise an HMGB A box.
In another embodiment, the HMGB B box is a polypeptide that is about 90%,
80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, or 20%, of the length of a full length
HMGB 1 polypeptide. In another embodiment, the HMGB B box comprises or
consists of the sequence of SEQ ID NO:S, SEQ ID N0:20 or SEQ ID N0:58, or the
amino acid sequence in the corresponding region of an HMGB protein in a
mammal,
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but is still less than the full length HMGB polypeptide. An HMGB B box
polypeptide is also a recombinantly produced polypeptide having the same amino
acid sequence as an HMGB B box polypeptide described above. Preferably, the
HMGB B box is a mammalian HMGB B box, for example, a human HMGB 1 B box.
An HMGB B box often has no more than about 85 amino acids and no fewer than
about 4 amino acids. Examples of polypeptides having B box sequences within
them
include, but are not limited to, the HMGB proteins and polypeptides described
herein. The B box sequences in such polypeptides can be determined and
isolated
using methods described herein, for example, by sequence comparisons to B
boxes
described herein and testing for biological activity, using methods described
herein or
other methods known in the art.
Additional examples of HMGB B box polypeptide sequences include the
following sequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(human HMGBl; SEQ ID NO: 51); KKDPNAPKRP PSAFFLFCSE HRPKIKSEHP
GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY (human
HMGB2; SEQ ID NO: 52); FKDPNAPKRL PSAFFLFCSE YRPKIKGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(HMG1L5 (formerly HMG1L10); SEQ ID NO: 53); FKDPNAPKRP PSAFFLFCSE
YHPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPGEKKA
AKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 54); FKDSNAPKRP
PSAFLLFCSE YCPKIKGEHP GLPISDVAKK LVEMWNNTFA DDKQLCEKKA
AKLKEKYKKD TATY (HMG1L4; SEQ ID NO: 55); FKDPNAPKRP
PSAFFLFCSE YRPKIKGEHP GLSIGDVVKK LAGMWNNTAA ADKQFYEKKA
AKLKEKYKKD IAAY (HMG sequence from BAC clone RP11-359A23; SEQ ID
NO: 56); and FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK
LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAYRAKGKP
DAAKKGVVKA EK (human HMGB 1 box; SEQ ID NO: 58).
The HMGB B box polypeptides of the invention also encompass sequence
variants. Variants include a substantially homologous polypeptide encoded by
the
same genetic locus in an organism, i.e., an allelic variant, as well as other
variants.
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Variants also encompass polypeptides derived from other genetic loci in an
organism, but having substantial homology to a polypeptide encoded by an HMGB
box nucleic acid molecule, and complements and portions thereof, or having
substantial homology to a polypeptide encoded by a nucleic acid molecule
comprising the nucleotide sequence of an HMGB B box nucleic acid molecule.
Examples of HMGB B box nucleic acid molecules are known in the art and can be
derived from HMGB B box polypeptides as described herein. Variants also
include
polypeptides substantially homologous or identical to these polypeptides but
derived
from another organism, i.e., an ortholog. Variants also include polypeptides
that are
substantially homologous or identical to these polypeptides that are produced
by
chemical synthesis. Variants also include polypeptides that are substantially
homologous or identical to these polypeptides that are produced by recombinant
methods. Preferably, a non-naturally occurring HMGB B box polypeptide has at
least 60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, and most
preferably at least 95%, sequence identity to the sequence of an HMGB B box as
described herein, for example, the sequence of SEQ ID NO:S, SEQ ID NO:20, or
SEQ ID N0:58, as determined using the BLAST program and parameters described
herein. Preferably, the HMGB B box consists of the sequence of SEQ ID NO:S,
SEQ
ID N0:20, or SEQ ID N0:58, or the amino acid sequence in the corresponding
region of an HMGB protein in a mammal, and has one or more of the biological
activities of an HMGB B box, as determined using methods described herein or
other
methods known in the art.
In other embodiments, the present invention is directed to a polypeptide
comprising an HMGB B box biologically active fragment that has B box
biological
activity, or a non-naturally occurring HMGB B box fragment In another
embodiment, the present invention is directed to a polypeptide comprising a
vertebrate HMGB B box or a fragment thereof that has B box biological
activity, or a
non-naturally occurring HMGB B box but not comprising a full length HMGB
polypeptide. By a "B box fragment that has B box biological activity" or a "B
box
biologically active fragment" is meant a fragment of an HMGB B box that has
the
activity of an HMGB B box. For example, the B box fragment can induce release
of
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a pro-inflammatory cytolcine from a vertebrate cell or increase inflammation,
or
induce the inflammatory cytolcine cascade. An example of such a B box fragment
is
the fragment comprising the first 20 amino acids of the HMGB 1 B box (SEQ ID
N0:16 or SEQ ID N0:23), as described herein. B box fragments can be generated
using standard molecular biology techniques and assaying the function of the
fragment by determining if the fragment, when administered to a cell,
increases
release of a proinflaxnmatory cytolcine from the cell, as compared to a
suitable
control, for example, using methods described herein or other methods known in
the
art.
HMGB polypeptides, HMGB A boxes, and HMGB B boxes, either naturally
occurring or non-naturally occuiTing, include polypeptides that have sequence
identity to the HMGB polypeptides, HMGB A boxes, and HMGB B boxes described
herein. As used herein, two polypeptides (or a region of the polypeptides) are
substantially homologous or identical when the amino acid sequences are at
least
about 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more, homologous or identical.
The percent identity of two amino acid sequences (or two nucleic acid
sequences)
can be determined by aligning the sequences for optimal comparison purposes
(e.g.,
gaps can be introduced in the sequence of a first sequence). The amino acids
or
nucleotides at corresponding positions are then compared, and the percent
identity
between the two sequences is a function of the number of identical positions
shared
by the sequences (i.e., % identity = # of identical positions/total # of
positions x 100).
In certain embodiments, the length of the HMGB polypeptide, HMGB A box
polypeptide, or HMGB B box polypeptide aligned for comparison purposes is at
least
30%, preferably, at least 40%, more preferably, at least 60%, and even more
preferably, at least 70%, 80%, 90%, or 100%, of the length of the reference
sequence,
for example, those sequence provided in FIGS. 12A-12E, FIGS. 14A-14P, and SEQ
ID NOS: 18, 20, and 22. The actual comparison of the two sequences can be
accomplished by well-known methods, for example, using a mathematical
algorithm.
A preferred, non-limiting example of such a mathematical algorithm is
described in
Karlin et al. (Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). Such an
algorithm
is incorporated into the BLASTN and BLASTX programs (version 2.2) as described
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in Schaffer et al. (Nucleic Acids Res., 29:2994-3005, 2001). When utilizing
BLAST
and Gapped BLAST programs, the default parameters of the respective programs
(e.g., BLASTN) can be used. See the Internet site for the National Center for
Biotechnology Information (NCBI). In one embodiment, the database searched is
a
non-redundant (NR) database, and parameters for sequence comparison can be set
at:
no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and
Gap
Costs have an Existence of 11 and an Extension of 1.
Another preferred, non-limiting example of a mathematical algorithm utilized
for the comparison of sequences is the algorithm of Myers and Miller, CABIOS
(1989). Such an algorithm is incorporated into the ALIGN program (version
2.0),
which is part of the GCG (Accelrys) sequence alignment software package. When
utilizing the ALIGN program for comparing amino acid sequences, a PAM120
weight residue table, a gap length penalty of 12 , and a gap penalty of 4 can
be used.
Additional algorithms for sequence analysis are known in the art and include
ADVANCE and ADAM as described in Torellis and Robotti (Comput. Appl. Biosci.,
10: 3-5,1994); and FASTA described in Pearson and Lipman (Proc. Natl. Acad.
Sci
USA, 85: 2444-2448, 1988).
In another embodiment, the percent identity between two amino acid
sequences can be accomplished using the GAP program in the GCG software
package (Accelrys, San Diego, California) using either a Blossom 63 matrix or
a
PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of
2, 3, or
4. In yet another embodiment, the percent identity between two nucleic acid
sequences can be accomplished using the GAP program in the GCG software
package (Accelrys, San Diego, California), using a gap weight of 50 and a
length
weight of 3.
As used herein, a "cytokine" is a soluble protein or peptide which is
naturally
produced by mammalian cells and which acts in vivo as a humoral regulator at
micro-
to picomolar concentrations. Cytoleines can, either under normal or
pathological
conditions, modulate the functional activities of individual cells and
tissues. A
proinflammatory cytolcine is a cytokine that is capable of causing any of the
following physiological reactions associated with inflammation: vasodilation,
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hyperemia, increased permeability of vessels with associated edema,
accumulation of
granulocytes and mononuclear phagocytes, or deposition of fibrin. In some
cases, the
proinflaxnmatory cytokine can also cause apoptosis, such as in chronic heart
failure,
where TNF has been shown to stimulate cardiomyocyte apoptosis (Pulkki, Ann.
Med. 29: 339-343, 1997; and Tsutsui et al., Immunol. Rev. 174:192-209, 2000).
Nonlimiting examples of proinflammatory cytokines are tumor necrosis
factor (TNF), interleulcin (IL)-la, IL-1(3, IL-6, IL-8, IL-18, interferon y,
HMG-l,
platelet-activating factor (PAF), and macrophage migration inhibitory factor
(MIF).
Proinflaxnmatory cytokines are to be distinguished from anti-inflammatory
cytokines, such as IL-4, IL-10, and IL-13, which are not mediators of
inflammation.
In many instances, proinflammatory cytokines are produced in an
inflammatory cytokine cascade, defined herein as an in vivo release of at
least one
proinflammatory cytokine in a mammal, wherein the cytokine release affects a
physiological condition of the mammal. Thus, an inflammatory cytokine cascade
is
inhibited in embodiments of the invention where proinflammatory cytokine
release
causes a deleterious physiological condition.
As used herein, "an agent that inhibits TNF biological activity" is an agent
that decreases one or more of the biological activities of TNF. Examples of
TNF
biological activity include, but are not limited to, vasodilation, hyperemia,
increased
permeability of vessels with associated edema, accumulation of granulocytes
and
mononuclear phagocytes, and deposition of fibrin. Agents that inhibit TNF
biological activity include agents that inhibit (decrease) the interaction
between TNF
and a TNF receptor. Examples of such agents include antibodies or antigen
binding
fragments thereof that bind to TNF, antibodies or antigen binding fragments
that bind
a TNF receptor, and molecules that bind TNF or the TNF receptor and prevent
TNF/TNF receptor interaction. Such agents include, but are not limited to
peptides,
proteins, synthesized molecules, for example, synthetic organic molecules,
naturally-occurring molecule, for example, naturally occurring organic
molecules,
nucleic acid molecules, and components thereof. Preferred examples of agents
that
inhibit TNF biological activity include infliximab (Remicade; Centocor, Inc.,
Malvern, Pennsylvania), etanercept (Immunex; Seattle, Washington), adalimumab
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(D2E7; Abbot Laboratories, Abbot Park Illinois), CDP870 (Pharmacia
Corporation;
Bridgewater, New Jersey) CDP571 (Celltech Group plc, United Kingdom),
Lenercept (Roche, Switzerland), and Thalidomide.
Inflammatory cytokine cascades contribute to deleterious characteristics,
including inflammation and apoptosis, of numerous disorders. Included are
disorders
characterized by both localized and systemic reactions, including, without
limitation,
the disorders described herein (e.g., those conditions enumerated in the
background
section of this specification). Particular disorders characterized by
inflammatory
cytolcine cascades include, e.g., sepsis, allograft rejection, rheumatoid
arthritis,
asthma, lupus, adult respiratory distress syndrome, chronic obstructive
pulmonary
disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia,
organic
ischemia, reperfusion ischemia, Behcet's disease, graft versus host disease,
Crohn's
disease, ulcerative colitis, multiple sclerosis, and cachexia.
A Box Polypeptides aid Biologically Active Ff°agments Thereof
As described herein, in one aspect the present invention is directed to a
polypeptide composition comprising a vertebrate HMGB A box, or a biologically
active fragment thereof, which can inhibit release of a proinflammatory
cytokine
from a cell treated with HMG, or which can be used to treat a condition
characterized
by activation of an inflarmnatory cytokine cascade. In certain embodiments,
the
invention is directed to compositions comprising an HMGB A box, or a
biologically
active fragment or variant thereof, in combination with one or more agents
that
inhibit TNF biological activity, for example, infliximab, etanercept,
adalimumab,
CDP870, CDP571, Lenercept, or Thalidomide. Such compositions can be used to
inhibit release of a proinflammatory cytokine from a vertebrate cell treated
with
HMG, and/or can be used to treat a condition characterized by activation of an
inflammatory cytokine cascade.
When referring to the effect of any of the compositions or methods of the
invention on the release of proinflammatory cytokines, the use of the terms
"inhibit"
or "decrease" encompasses at least a small but measurable reduction in
proinflammatory cytolcine release. In preferred embodiments, the release of
the
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proinflammatory cytokine is inhibited by at least 20% over non-treated
controls; in
more preferred embodiments, the inlubition is at least 50%; in still more
preferred
embodiments, the inhibition is at least 70%, and in the most preferred
embodiments,
the inhibition is at least SO%. Inhibition can be assessed using methods
described
herein or other methods known in the art. Such reductions in proinflammatory
cytokine release are capable of reducing the deleterious effects of an
inflammatory
cytokine cascade in i~ vivo embodiments.
Because HMGB A boxes (e.g., vertebrate HMGB A boxes) show a high
degree of sequence conservation (see, for example, FIG. 13 for an amino acid
sequence comparison of rat, mouse, and human HMGB polypeptides), it is
believed
that an HMGB A box (e.g., a vertebrate HMGB A box) can inhibit release of a
proinflammatory cytokine from a vertebrate cell treated with HMGB. Therefore,
an
HMGB A box (e.g., a vertebrate HMGB A box) is within the scope of the
invention.
Preferably, the HMGB A box is a vertebrate HMGB A box (e.g., a mammalian
HMGB A box, such as a human HMGB 1 A box provided herein as SEQ ID N0:4,
SEQ ID N0:22, or SEQ ID N0:57). Also included in the present invention are
fragments of the HMGB 1 A box having HMGB A box biological activity, as
described herein.
It would also be recognized by the spilled artisan that non-naturally
occurring
HMGB A boxes (or biologically active fragments thereof) can be created without
undue experimentation, which would inhibit release of a proinflammatory
cytolcine
from a vertebrate cell treated with a vertebrate HMGB. These non-naturally
occurring functional A boxes (variants) can be created by aligning amino acid
sequences of HMGB A boxes from different sources, and mal~ing one or more
substitutions in one of the sequences at amino acid positions where the A
boxes
differ. The substitutions are preferably made using the same amino acid
residue that
occurs in the compared A box. Alternatively, a conservative substitution is
made
from either of the residues.
Conservative amino acid substitutions refer to the interchangeability of
residues having similar side chains. Conservatively substituted amino acids
can be
grouped according to the chemical properties of their side chains. For
example, one
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grouping of amino acids includes those amino acids have neutral and
hydrophobic
side chains (a, v, l, i, p, w, f, and m); another grouping is those amino
acids having
neutral and polar side chains (g, s, t, y, c, n, and q); another grouping is
those amino
acids having basic side chains (lc, r, and h); another grouping is those amino
acids
having acidic side chains (d and e); another grouping is those amino acids
having
aliphatic side chains (g, a, v, l, and i); another grouping is those amino
acids having
aliphatic-hydroxyl side chains (s and t); another grouping is those amino
acids having
amine-containing side chains (n, q, k, r, and h); another grouping is those
amino
acids having aromatic side chains (f, y, and w); and another grouping is those
amino
acids having sulfur-containing side chains (c and m). Preferred conservative
amino
acid substitutions groups are: r-k; e-d, y-f, l-m; v-i, and q-h.
While a conservative amino acid substitution would be expected to preserve
the biological activity of an HMGB A box polypeptide, the following is one
example
of how non-naturally occurring A box polypeptides (variants) can be made by
comparing the human HMGB 1 A box (SEQ ID NO:4) with residues 32 to 85 of SEQ
ID NO:3 of the human HMGB2 A box (SEQ ID N0:17).
HMGB 1 pdasvnfsef sklccserwkt msakekgkfe dmakadkary eremktyipp kget (SEQ ID
N0:4)
HMGB2 pdssvnfaef sklccserwkt msakekskfe dmaksdkary dremlcnyvpp kgdk (SEQ ID
N0:17)
A non-naturally occmTing HMGB A box can be created by, for example, by
substituting the alanine (a) residue at the third position in the HMGBl A box
with
the serine (s) residue that occurs at the third position of the HMGB2 A box.
The
skilled artisan would lcnow that the substitution would provide a functional
non-
naturally occurring A box because the s residue functions at that position in
the
HMGB2 A box. Alternatively, the third position of the HMGB 1 A box can be
substituted with any amino acid that is conservative to alanine or serine,
such as
glycine (g), threonine (t), valine (v) or leucine (1). The skilled artisan
would
recognize that these conservative substitutions would be expected to result in
a
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functional A box because A boxes are not invariant at the third position, so a
conservative substitution would provide an adequate structural substitute for
an
amino acid that is naturally occurring at that position.
Following the above method, a great many non-naturally occurring HMGB A
boxes could be created without undue experimentation wluch would be expected
to
be functional, and the functionality of any particular non-naturally occurring
HMGB
A box could be predicted with adequate accuracy. In any event, the
functionality of
any non-naturally occurring HMGB A box could be determined without undue
experimentation by simply adding it to cells along with an HMGB polypeptide,
and
determining whether the A box inhibits release of a proinflammatory cytokine
by the
cells, using, for example, methods described herein.
The cell from which the A box or an A box biologically active fragment will
inhibit the release of HMG-induced proinflammatory cytokines can be any cell
that
can be induced to produce a proinflammatory cytokine. In preferred
embodiments,
the cell is a mammalian cell, for example, an immune cell (e.g., a macrophage,
a
monocyte, or a neutrophil).
Polypeptides comprising an A box or A box biologically active fragment that
can inhibit the production of any single proinflarnmatoiy cytokine, now known
or
later discovered, are within the scope of the present invention. Preferably,
the
antibodies can inhibit the production of TNF, IL-1 Vii, and/or IL-6. Most
preferably,
the antibodies can inhibit the production of any proinflammatory cytokines
produced
by the vertebrate cell.
B Box Polypeptides and Biologically Active Ff~agrnents Thereof
As described herein, in one aspect the present invention is directed to a
polypeptide composition comprising a vertebrate HMGB B box, or a biologically
active fragment thereof, which can increase release of a proinflammatory
cytokine
from a vertebrate cell treated with HMGB.
When referring to the effect of any of the compositions or methods of the
invention on the release of proinflammatory cytokines, the use of the term
"increase"
encompasses at least a small but measurable rise in proinflammatory cytokine
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release. In preferred embodiments, the release of the proinflammatory cytokine
is
increased by at least 1.5-fold, at least 2-fold, at least 5-fold, or at least
10-fold, over
non-treated controls. Such increases in proinflammatory cytolcine release are
capable
of increasing the effects of an inflammatory cytokine cascade in in vivo
embodiments. Such polypeptides can also be used to induce weight loss and/or
treat
obesity.
Because all HMGB B boxes show a high degree of sequence conservation
(see, for example, FIG. 13 for an amino acids sequence comparison of rat,
mouse,
and human HMGB polypeptides), it is believed that functional non-naturally
occurring HMGB B boxes can be created without undue experimentation by making
one or more conservative amino acid substitutions, or by comparing naturally
occurring vertebrate B boxes from different sources and substituting analogous
amino acids, as was discussed above with respect to the creation of functional
non-
naturally occurring A boxes. In particularly preferred embodiments, the B box
comprises SEQ ID NO:S, SEQ ID NO: 20 or SEQ ID NO:58, which are the
sequences (three different lengths) of the human HMGB 1 B box, or, comprises
the B
box sequences from the polypeptides shown in FIGS. 14A-14P, or is a fragment
of
an HMGB B box that has B box biological activity. For example, a 20 amino acid
sequence contained within SEQ ID NO: 20 contributes to the function of the B
box.
This 20 amino acid B-box fragment has the following amino acid sequence:
flcdpnapkrl psafflfcse (SEQ ID N0:23). Another example of an HMGB B box
biologically active fragment consists of amino acids 1-20 of SEQ ID NO:S
(naplcrppsaf flfcseyrplc; SEQ ID NO: 16).
Antibodies to HMGB and HMGB B Box Polypeptides
The invention is also directed to a purified preparation of antibodies that
bind
to an HMGB polypeptide or a biologically active fragment thereof (anti-HMGB
antibodies). The anti-HMGB antibodies can be neutralizing antibodies (i.e.,
can
inhibit a biological activity of an HMG polypeptide or a biologically active
fragment
thereof, for example, the release of a proinflammatory cytokine from a
vertebrate cell
induced by HMG). The invention is also directed to a purified preparation of
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antibodies that specifically bind to a vertebrate high mobility group protein
(HIVIG) B
box or a biologically active fragment thereof, but do not selectively bind to
non-B
box epitopes of HMGB (anti-HMGB B box antibodies). In these embodiments, the
antibodies can also be neutralizing antibodies (i.e., they can inhibit a
biological
activity of a B box polypeptide or biologically active fragment thereof, for
example,
the release of a proinflammatory cytokine from a vertebrate cell induced by
HMGB).
Such antibodies can be combined with one or more agents that inhibit TNF
biological activity, for example, infliximab, etanercept, adalimumab, CDP870,
CDP571, Lenercept, or Thalidomide.
The term "antibody" or "purified antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site that
selectively binds
an antigen. A molecule that selectively binds to a polypeptide of the
invention is a
molecule that binds to that polypeptide or a fragment thereof, but does not
substantially bind other molecules in a sample, e.g., a biological sample that
naturally
contains the polypeptide. Preferably the antibody is at least 60%, by weight,
free
from proteins and naturally occurring organic molecules with which it is
naturally
associated. More preferably, the antibody preparation is at least 75% or 90%,
and
most preferably, 99%, by weight, antibody. , Examples of immunologically
active
portions of immunoglobulin molecules include Flab) and F(ab')Z fragments that
can
be generated by treating the antibody with an enzyme such as pepsin.
The invention provides polyclonal and monoclonal antibodies that selectively
bind to a HMGB B box polypeptide of the invention. The term "monoclonal
antibody" or "monoclonal antibody composition," as used herein, refers to a
population of antibody molecules that contain only one species of an antigen
binding
site capable of immunoreacting with a particular epitope of a polypeptide of
the
invention. A monoclonal antibody composition thus typically displays a single
binding affinity for a particular polypeptide of the invention with which it
immunoreacts.
Polyclonal antibodies can be prepared, e.g., as described herein, by
immunizing a suitable subject with a desired immunogen, e.g., an HMGB B box
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polypeptide of the invention or fragment thereof. The antibody titer in the
immunized subject can be monitored over time by standard techniques, such as
with
an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If
desired, the antibody molecules directed against the polypeptide can be
isolated from
the mammal (e.g., from the blood) and further purified by well-known
techniques,
such as protein A chromatography to obtain the IgG fraction.
At an appropriate time after immunization, e.g., when the antibody titers are
highest, antibody-producing cells can be obtained from the subject and used to
prepare monoclonal antibodies by standard techniques, such as the hybridoma
technique originally described by Kohler and Milstein (Nature 256:495-497,
1975),
the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72,
1983),
the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or trioma techniques. The
technology
for producing hybridomas is well known (see generally Current Protocols in
Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY,
1994).
Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a marmnal immunized with an immunogen as
described
above, and the culture supernatants of the resulting hybridoma cells are
screened to
identify a hybridoma producing a monoclonal antibody that binds a particular
polypeptide (e.g., a polypeptide of the invention).
Any of the marry well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating a
monoclonal
antibody to a polypeptide of the invention (see, e.g., Current Protocols in
Immunology, supra; Galfre et al. (Nature, 266:55052, 1977); R.H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum
Publishing Corp., New York, New York (1980); and Lerner (Yale J. Biol. Med.
54:387-402, 1981)). Moreover, the ordinarily skilled worker will appreciate
that
there are many variations of such methods that also would be useful.
In one alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody to an HMGB B box polypeptide of the invention can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin
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library (e.g., an antibody phage display library) with the polypeptide to
thereby
isolate immunoglobulin library members that bind the polypeptide. Kits for
generating and screening phage display libraries are commercially available
(e.g., the
Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-O1; and the
Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in generating
and
screening antibody display library can be found in, for example, U.S. Patent
No.
5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271;
PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT
Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT
Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al.,
BiofTechnology 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-
85,
1992; Huse et al. (Science 246:1275-1281, 1989); and Griffiths et al. (EMBO J.
12:725-734, 1993).
Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made using standard recombinant DNA techniques, are within the scope of the
invention. Such chimeric and humanized monoclonal antibodies can be produced
by
recombinant DNA techniques known in the art.
In general, antibodies of the invention (e.g., a monoclonal antibody) can be
used to isolate an HMGB B box polypeptide of the invention by standard
techniques,
such as affinity chromatography or immunoprecipitation. A polypeptide-specific
antibody can facilitate the purification of natural polypeptide from cells and
of
recombinantly produced polypeptide expressed in host cells. Moreover, an
antibody
specific for an HMGB B box polypeptide of the invention can be used to detect
the
polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample)
in order to
evaluate the abundance and pattern of expression of the polypeptide.
Because vertebrate HMGB polypeptides and HMGB B boxes show a high
degree of sequence conservation, it is believed that vertebrate HMGB
polypeptides or
HMGB B boxes in general can induce release of a proinflammatory cytokine from
a
vertebrate cell. Therefore, antibodies against vertebrate HMGB polypeptides or
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HMGB B boxes are within the scope of the invention. In one embodiment, the
antibodies are neutralizing antibodies.
Preferably, the HMGB polypeptide is a mammalian HMG, as described
herein, more preferably a mammalian HMGB 1 polypeptide, most preferably a
human
HMGB 1 polypeptide, provided herein as SEQ ID NO:1. Antibodies can also be
directed against an HMGB polypeptide fragment that has HMGB polypeptide
biological activity.
Preferably, the HMGB B box is a mammalian HMGB B box, more preferably
a mammalian HMGB 1 B box, most preferably a human HMGB 1 B box, provided
herein as SEQ ID NO:S, SEQ ID N0:20, or SEQ ID N0:58. Antibodies can also be
directed against an HMGB B box fragment that has B box biological activity.
Antibodies generated against an HMGB immunogen or an HMGB B box
immunogen can be obtained by administering an HMGB polypeptide, or fragment
thereof, an HMGB B box or fragment thereof, or cells comprising the HMGB
polypeptide, the HMGB B box, or fragments thereof, to an animal, preferably a
nonhuman, using routine protocols. The polypeptide, such as an antigenically
or
immunologically equivalent derivative, is used as an antigen to immunize a
mouse or
other animal, such as a rat or chicken. The immunogen may be associated, for
example, by conjugation, with an immunogenic carrier protein, for example,
bovine
serum albumin (BSA) or lceyhole limpet haemocyanin (KLH). Alternatively, a
multiple antigenic peptide comprising multiple copies of the HMGB or HMGB B
box or fragment, may be sufficiently antigenic to improve immunogenicity so as
to
obviate the need for a carrier. Bispecific antibodies, having two antigen
binding
domains where each is directed against a different HMGB or HMGB B box epitope,
may also be produced by routine methods.
For preparation of monoclonal antibodies, any technique known in the art that
provides antibodies produced by continuous cell line cultures can be used.
See, e.g.,
Kohler and Milstein, supra; and Cole et al., supra.
Techniques for the production of single chain antibodies (LJ.S. Pat. No.
4,946,778) can be adapted to produce single chain antibodies to HMGB, the B
box or
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fragments thereof. Also, transgenic mice, or other organisms such as other
mammals, may be used to express humanized antibodies.
If the antibody is used therapeutically in ih vivo applications, the antibody
is
preferably modified to make it less immunogenic in the individual. For
example, if
the individual is human the antibody is preferably "humanized"; where the
complementarity determining regions) of the antibody is transplanted into a
human
antibody (for example, as described in Jones et al. (Nature 321:522-525,
1986); and
Tempest et al. (Biotechnology 9:266-273, 1991)).
Phage display technology can also be utilized to select antibody genes with
binding activities towards the polypeptide either from repertoires of PCR
amplified
v-genes of lymphocytes from humans screened for possessing anti-B box
antibodies
or from naive libraries (McCafferty et al., Nature 348:552-554, 1990; and
Marks, et
al., Biotechnology 10:779-783, 1992). The affinity of these antibodies can
also be
improved by chain shuffling (Clackson et al., Nature 352: 624-628, 1991).
When the antibodies are obtained that specifically bind to HMGB epitopes or
to HMGB B box epitopes, they can then be screened, without undue
experimentation, for the ability to inhibit release of a proinflammatory
cytokine.
Anti-HMGB B box antibodies that can inhibit the production of any single
proinflammatory cytolcine, and/or inhibit the release of a proinflammatory
cytokine
from a cell, and/or inhibit a condition characterized by activation of an
inflammatory
cytolcine cascade, are within the scope of the present invention. Preferably,
the
antibodies can inhibit the production of TNF, IL-1 (3, and/or IL-6. Most
preferably,
the antibodies can inhibit the production of any proinflammatory cytokines
produced
by the vertebrate cell.
For methods of inhibiting release of a proinflammatory cytolcine from a cell
or treating a condition characterized by activation of an inflammatory
cytokine
cascade using antibodies to the HMGB B box or a biologically active fragment
thereof, the cell can be any cell that can be induced to produce a
proinflammatory
cytolcine. In preferred embodiments, the cell is an immune cell, for example,
macrophages, monocytes, or neutrophils.
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Compositions Comprising One or More of an HMGB A box polypeptide, an Antibody
to HMGB, an Aratibody to an HMGB B box, and an Inhibitor of .TNFBiological
Activity
In certain embodiments, the present invention is directed to a composition
comprising any of the above-described polypeptides (e.g., an HMGB A box
polypeptide or biologically active fragment as described herein) in a
pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable
carriers
include those described herein. In these embodiments, the composition can
inhibit a
condition characterized by activation of an inflammatory cytokine cascade. The
condition can be one where the inflammatory cytokine cascade causes a systemic
reaction, such as with endotoxic shocle. Alternatively, the condition can be
mediated
by a localized inflammatory cytokine cascade, as in rheumatoid arthritis:
Nonlimiting examples of conditions which can be usefully treated using the
present
invention include those conditions enumerated in the background section of
this
specification. In one embodiment, the condition to be treated is appendicitis,
peptic,
gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative,
pseudomembranous,
acute or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy,
anaphylactic
shoclc, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis,
septicemia, endotoxic shock, cachexia, septic abortion, disseminated
bacteremia,
bums, Alzheimer's disease, coeliac disease, congestive heart failure, adult
respiratory
distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury,
paralysis, allograft rejection or graft-versus-host disease. In another
embodiment, the
condition is endotoxic shock or allograft rej ection. Where the condition is
allograft
rejection, the composition may advantageously also include an
immunosuppressant
that is used to inhibit allograft rejection, such as cyclosporin.
In other embodiments, the invention is directed to a composition comprising
the antibody preparations described above (e.g., anti-HMGB B box antibodies or
biologically active fragments thereof, as described herein), in a
pharmaceutically
acceptable caxrier. In these embodiments, the compositions can intubit a
condition
characterized by the activation of an inflammatory cytokine cascade.
Conditions that
can be treated with these compositions have been previously enumerated.
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In other embodiments, the invention is directed to a composition comprising
any of the above-described HMGB A box polypeptides, and/or an antibody or
antigen binding fragment thereof that binds HMGB, and/or an antibody or
antigen
binding fragment thereof that binds an HMGB B box, and an agent that inhibits
TNF
biological activity (collectively termed "combination therapy compositions").
Preferred examples of agents that inhibit TNF biological activity include
infliximab,
etanercept, adalimumab, CDP870, CDP571, Lenercept, and Thalidomide. Such
combination therapy compositions can further comprise a pharmaceutically
acceptable carrier. Suitable pharmaceutically acceptable carriers include
those .
described herein. In these embodiments, the combination therapy composition
can
inhibit a condition characterized by activation of an inflarmnatory cytokine
cascade
and/or inhibit release of a proinflammatoiy cytokine from a cell. The
condition can
be one where the inflammatory cytokine cascade causes a systemic reaction,
such as
with endotoxic shock. Alternatively, the condition can be mediated by a
localized
inflammatory cytokine cascade, as in rheumatoid arthritis. Nonlimiting
examples of
conditions which can be usefully treated using the present invention include
those
conditions enumerated in the background section of this specification. In one
embodiment, the condition to be treated is sepsis, allograft rejection,
rheumatoid
arthritis, asthma, lupus, adult respiratory distress syndrome, chronic
obstructive
pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial
ischemia,
organic ischemia, reperfusion ischemia, Behcet's disease, graft versus host
disease,
Crohn's disease, ulcerative colitis, multiple sclerosis, and cachexia.
Preferably the
combination therapy compositions are administered to a patient in need thereof
in an
amount sufficient to inhibit release of proinflammatory cytolcine from a cell
and/or to
treat a condition characterized by activation of an inflammatory cytolcine
cascade. In
one embodiment, release of the proinflammatory cytokine is inhibited by at
least
10%, 20%, 25%, 50%, 75%, 80%, 90% or 95%, as assessed using methods described
herein or other methods lcnown in the art.
The carrier or excipient included with the polypeptide (e.g., an HMGB A box
polypeptide or biologically active fragment thereof), antibody (e.g., an anti-
HMGB B
box antibody or biologically active fragment thereof) or combination therapy
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composition (e.g., an HMGB A box polypeptide or biologically active fragment
thereof and an agent that inhibits TNF biological activity, and/or an antibody
or
antigen binding fragment thereof that binds HMGB and an agent that inhibits
TNF
biological activity, and/or an antibody or antigen binding fragment thereof
that binds
an HMGB B box and an agent that inhibits TNF biological activity) is chosen
based
on the expected route of administration of the composition in therapeutic
applications. The route of administration of the composition depends on the
condition to be treated. For example, intravenous injection may be preferred
for
treatment of a systemic disorder such as endotoxic shock, and oral
administration
may be preferred to treat a gastrointestinal disorder such as a gastric ulcer.
The route
of administration and the dosage of the composition to be administered can.be
determined by the skilled artisan, without undue experimentation, in
conjunction
with standard dose-response studies. Relevant circumstances to be considered
in
making such determinations include the condition or conditions to be treated,
the
choice of composition to be administered, the age, weight, and response of the
individual patient, and the severity of the patient's symptoms. Thus,
depending on
the condition, the antibody composition can be administered orally,
parenterally,
intranasally, vaginally, rectally, lingually, sublingually, bucally,
intrabuccaly and
transdermally to the patient.
Accordingly, compositions designed for oral, lingual, sublingual, buccal and
intrabuccal administration can be made without undue experimentation by means
well known in the art, for example, with an inert diluent or with an edible
carrier.
The compositions may be enclosed in gelatin capsules or compressed into
tablets.
For the purpose of oral therapeutic administration, the pharmaceutical
compositions
of the present invention may be incorporated with excipients and used in the
form of
tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums
and the
like.
Tablets, pills, capsules, troches and the like may also contain binders,
recipients, disintegrating agent, lubricants, sweetening agents, and flavoring
agents.
Some examples of binders include microcrystalline cellulose, gum tragacanth
and
gelatin. Examples of excipients include starch and lactose. Some examples of
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disintegrating agents include alginic acid, corn starch and the like. Examples
of
lubricants include magnesium stearate and potassium stearate. An example of a
glidant is colloidal silicon dioxide. Some examples of sweetening agents
include
sucrose, saccharin and the like. Examples of flavoring agents include
peppermint,
methyl salicylate, orange flavoring and the like. Materials used in preparing
these
various compositions should be pharmaceutically pure and non-toxic in the
amounts
used.
The compositions of the present invention can easily be administered
parenterally such as, for example, by intravenous, intramuscular, intrathecal
or
subcutaneous injection. Parenteral administration can be accomplished by
incorporating the compositions of the present invention into a solution or
suspension.
Such solutions or suspensions may also include sterile diluents such as water
for
injection, saline solution, fixed oils, polyethylene glycols, glycerine,
propylene glycol
andlor other synthetic solvents. Parenteral formulations may also include
antibacterial agents such as, for example, benzyl alcohol and/or methyl
parabens,
antioxidants such as, for example, ascorbic acid and/or sodium bisulfate and
chelating
agents such as EDTA. Buffers, such as acetates, citrates and/or phosphates,
and
agents for the adjustment of tonicity, such as sodium chloride and/or
dextrose, may
also be added. The parenteral preparation can be enclosed in ampules,
disposable
syringes or multiple dose vials made of glass or plastic.
Rectal administration includes administering the pharmaceutical
compositions into the rectum or large intestine. This can be accomplished
using
suppositories or enemas. Suppository formulations can easily be made by
methods
known in the art. For example, suppository formulations can be prepared by
heating
glycerin to about 120°C, dissolving the polypeptide composition,
antibody
composition and/or combination therapy composition in the glycerin, mixing the
heated glycerin after which purified water may be added, and pouring the hot
mixture
into a suppository~mold.
Transdermal administration includes percutaneous absorption of the
composition through the skin. Transdermal formulations include patches,
ointments,
creams, gels, salves and the like.
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The present invention includes nasally administering to a mammal (e.g., a
human) a therapeutically effective amount of the composition. As used herein,
nasally administering or nasal administration includes administering the
composition
to the mucous membranes of the nasal passage or nasal cavity of the patient.
As used
herein, pharmaceutical compositions for nasal administration of a composition
include therapeutically effective amounts of the polypeptide, antibody and/or
combination therapy agents, prepared by well-known methods to be administered,
for
example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or
powder.
Administration of the composition may also take place using a nasal tampon or
nasal
sponge.
The pharmaceutical compositions (e.g., polypeptide compositions, antibody
compositions and/or combination therapy composition) described herein can also
include an antagonist of an early sepsis mediator. As used herein, an early
sepsis
mediator is a proinflammatory cytokine that is released from cells soon (i.e.,
within
30-60 min.) after induction of an inflammatory cytokine cascade (e.g.,
exposure to
LPS). Nonlimiting examples of these cytokines axe TNF, IL-1 a, IL-1 (3, IL-6,
PAF,
and MIF. Also included as early sepsis mediators are receptors for these
cytokines
(for example, tumor necrosis factor receptor type 1) and enzymes required for
production of these cytokines, for example, interleukin-1 (3 converting
enzyme).
Antagonists of any early sepsis mediator, now known or later discovered, can
be
useful for these embodiments by further inhibiting an inflammatory cytokine
cascade.
Nonlimiting examples of antagonists of early sepsis mediators are antisense
compounds that bind to the mRNA of the early sepsis mediator, preventing its
expression (see, e.g., Ojwang et al. (Biochemistry 36:6033-6045, 1997);
Pampfer et
al. (Biol. Reprod. 52:1316-1326, 1995); U.S. Patent No. 6,228,642; Yahata et
al.
(Antisense Nucleic Acid Drug Dev. 6:55-61, 1996); and Taylor et al. (Antisense
Nucleic Acid Drug Dev. 8:199-205, 1998)), ribozymes that specifically cleave
the
mRNA of the early sepsis mediator (see, e.g., Leavitt et al. (Antisense
Nucleic Acid
Drug Dev. 10: 409-414, 2000); Hendrix et al. (Biochem. J. 314 (Pt. 2): 655-
661,
1996)), and antibodies that bind to the early sepsis mediator and inhibit
their action
(see, e.g., Kam and Targan (Expert Opin. Pharmacother. 1: 615-622, 2000);
Nagahira
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et al. (J. Immunol. Methods 222, 83-92, 1999); Lavine et al. (J. Cereb. Blood
Flow
Metab. 18: 52-58, 1998); and Holines et al. (Hybridoma 19: 363-367, 2000)).
Any
antagonist of an early sepsis mediator, now known or later discovered, is
envisioned
as within the scope of the invention. The skilled artisan can determine the
amount of
early sepsis mediator to use in these compositions for inhibiting any
particular
inflammatory cytokine cascade without undue experimentation with routine dose-
response studies.
Other agents that can be administered with the compositions described herein
include, e.g., Vitaxin~" and other antibodies targeting a~~33 integrin (see,
e.g., U.S.
Patent No. 5,753,230, PCT Publication Nos. WO 00/78815 and WO 02/070007; the
entire teachings of all of which are incorporated herein by reference) and
anti-IL-9
antibodies (see, e.g., PCT Publication No. WO 97/08321; the entire teachings
of
which are incorporated herein by reference). Additional agents that can be
administered with the polypeptide compositions described herein include, e.g.,
B7
antagonists (e.g., CTLA4Ig, anti-CD80 antibodies, anti-CD86 antibodies),
methotrexate, and/or CD40 antagonists (e.g., anti-CD40 ligand (CD40L)) (see,
e.g.,
Saito et al., J. Immunol. 160(9):4225-31 (1998)).
In further embodiments, the present invention is also directed to a method of
inhibiting the release of a proinflammatory cytokine from a mammalian cell.
The
method comprises treating the cell with any of the HMGB A box compositions,
and/or any of the HMGB B box or HMGB B box biologically active fragment
antibody compositions, and/or any of the combination therapy compositions
discussed above. It is believed that this method would be useful for
inhibiting the
cytokine release from any mammalian cell that produces a proinflarnlnatory
cytokine.
However, in preferred embodiments, the cell is a macrophage, because
macrophage
production of proinflammatory cytol~ines is associated with several important
diseases.
It is believed that this method is useful for the inhibition of any
proinflammatory cytokine produced by mammalian cells. In preferred
embodiments,
the proinflarnmatory cytokine is TNF, IL-1a, IL-1 Vii, MIF and/or IL-6,
because those
proinflammatory cytolcines are particularly important mediators of disease.
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The methods of these embodiments are useful for isa vitro applications, such
as in studies for determining biological characteristics of proinflammatory
cytol~ine
production in cells. However, the preferred embodiments are in vivo
therapeutic
applications, where the cells are in a patient suffering from, or at risk for,
a condition
characterized by activation of an inflammatory cytokine cascade.
In certain embodiments, the present invention is directed to a method of
treating a condition in a patient characterized by activation of an
inflammatory
cytokine cascade. The method comprises administering to the patient any of the
HMGB A box compositions (including non-naturally occurring A box polypeptides
and A box biologically active fragments), any of the HMGB B box or B box
biologically active fragment antibody compositions (including non-naturally
occurnng B box polypeptides or biologically.active fragments thereof), and/or
any of
the combination therapy compositions discussed above. This method would be
expected to be useful for any condition that is mediated by an inflammatory
cytokine
cascade, including any of those that have been previously enumerated. As with
previously described ifZ vivo methods, preferred conditions include
appendicitis,
peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative,
pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease,
asthma,
allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ
necrosis, hay
fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion,
disseminated
bacteremia, burns, Alzheimer's disease, cerebral infarction, cerebral
embolism, spinal
cord injury, paralysis, allograft rejection or graft-versus-host disease. In
the most
preferred embodiments, the condition is endotoxic shoclc or allograft
rejection.
Where the condition is allograft rejection, the composition may advantageously
also
include an immunosuppressant that is used to inhibit allograft rejection, such
as
cyclosporin.
These methods can also usefully include the administration of an antagonist
of an eaxly sepsis mediator, an anti-a~~i3 antibody, an anti IL-9 antibody, a
B7
antagonist (e.g., CTLA4Ig, an anti-CD80 antibody, an anti-CD86 antibody),
methotrexate, and/or a CD40 antagonist (e.g., anti-CD40 ligand (CD40L)). The
nature of these agents has been previously discussed.
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The B box polypeptides and biologically active fragments thereof described
herein can be used to induce inflammatory cytokines in the appropriate
isolated cells
in vitro, or ex vivo, or as a treatment ih vivo. In any of these treatments,
the
polypeptide or fragment can be administered by providing a DNA or RNA vector
encoding the B box or B box fragment, with the appropriate control sequences
operably linked to the encoded B box or B box fragment, so that the B box or B
box
fragment is synthesized in the treated cell or patient. he vivo applications
include the
use of the B box polypeptides or B box fragment polypeptides or vectors as a
weight
loss treatment. See WO 00/47104 (the entire teachings of which are
incorporated
herein by reference), demonstrating that treatment with HMGB 1 induces weight
loss.
In certain embodiments, the present invention is directed to methods of
stimulating the release of a proinflammatory cytokine from a cell. The method
comprises treating the cell with any of the B box polypeptides or biologically
active
B box fragment polypeptides, for example, polypeptides that comprise or
consist of
the sequence of SEQ ID NO:S, SEQ ID N0:20, SEQ ID NO:S~, SEQ ID N0:16, or
SEQ ID N0:23, as described herein (including non-naturally occurring B box
polypeptides and fragments). This method is useful for in vitro applications,
for
example, for studying the effect of proinflarmnatory cytokine production on
the
biology of the producing cell. Since the HMGB B box has the activity of the
HMGB
protein, the B box would also be expected to induce weight loss. Therefore, in
additional embodiments, the present invention is a method for effecting weight
loss
or treating obesity in a patient. The method comprises administering to the
patient an
effective amount of any of the B box polypeptides or B box fragment
polypeptides
described herein (including non-naturally occurring B box polypeptides and
fragments). In another embodiment, the B box polypeptide or B box fragment
polypeptide is in a pharmaceutically acceptable carrier.
Screening for Modulators of the Release of Proinflammatory Cytolcines from
Cells
The present invention is also directed to a method of determining whether a
compound (test compound) inhibits inflammation and/or an inflammatory
response.
The method comprises combining the compound with (a) a cell that releases a
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proinflammatory cytolcine when exposed to a vertebrate HMGB B box or a
biologically active fragment thereof, and (b) the HMGB B box or a biologically
active fragment thereof, and then determining whether the compound inhibits
the
release of the proinflammatory cytokine from the cell, as compared to a
suitable
control. A compound that inhibits the release of the proinflaxnmatory cytokine
in this
assay is a compound that can be used to treat inflammation and/or an
inflammatory
response. The HMGB B box or biologically active HMGB B box fragment can be
endogenous to the cell or can be introduced into the cell using standard
recombinant
molecular biology techniques.
Any cell that releases a proinflammatory cytokine in response to exposure to
a vertebrate HMGB B box or biologically active fragment thereof in the absence
of a
test compound would be expected to be useful for this invention. It is
envisioned
that the cell that is selected would be important in the etiology of the
condition to be
treated with the inhibitory compound that is being tested. For many
conditions, it is
expected that the preferred cell is a human macrophage.
Any method for determining whether the compound inhibits the release of the
proinflammatory cytokine from the cell would be useful for these embodiments.
It is
envisioned that the preferred methods are the direct measurement of the
proinflarmnatory cytolcine, for example, with any of a number of cormnercially
available ELISA assays. However, in some embodiments, the measurement of the
inflammatory effect of released cytokines may be preferable, particularly when
there
are several proinflammatory cytokines produced by the test cell. As previously
discussed, for many important disorders, the predominant proinflammatory
cytokines
axe TNF, IL-la, IL-lei, MIF or IL-6; particularly TNF.
The present invention also features a method of determining whether a
compound increases an inflammatory response and/or inflammation. The method
comprises combining the compound (test compound) with (a) a cell that releases
a
proinflammatory cytokine when exposed to a vertebrate HMGB A box or a
biologically active fragment thereof, and (b) the HMGB A box or biologically
active
fragment, and then determining whether the compound increases the release of
the
proinflammatory cytolcine from the cell, as compared to a suitable control. A
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compound that increases the release of the proinflammatory cytokine in this
assay is
a compound that can be used to increase an inflammatory response and/or
inflammation. The HMGB A box or HMGB A box biologically active fragment can
be endogenous to the cell or can be introduced into the cell using standard
recombinant molecular biology techniques.
Similar to the cell types useful for identifying inhibitors of inflammation
described above, any cell in which release of a proinflammatory cytokine is
normally
inhibited in response to exposure to a vertebrate HMGB A box or a biologically
active fragment thereof in the absence of any test compound would be expected
to be
useful for this invention. It is envisioned that the cell that is selected
would be
important in the etiology of the condition to be treated with the inhibitory
compound
that is being tested. For many conditions, it is expected that the preferred
cell is a
human macrophage.
Any method for determining whether the compound increases the release of
the proinflammatory cytokine from the cell would be useful for these
embodiments.
It is envisioned that the preferred methods are the direct measurement of the
proinflammatory cytokine, for example, with any of a number of commercially
available ELISA assays. However, in some embodiments, the measurement of the
inflammatory effect of released cytokines may be preferable, particularly when
there
are several proinflammatory cytokines produced by the test cell. As previously
discussed, for many important disorders, the predominant proinflammatory
cytokines
are TNF, IL-la, IL-lei, MIF or IL-6; particularly TNF.
Preferred embodiments of the invention are described in the following
examples. Other embodiments within the scope of the invention will be apparent
to
one skilled in the art from consideration of the specification or practice of
the
invention as disclosed herein. It is intended that the specification, together
with the
examples and claims, be considered exemplary only.
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Example 1: Materials and Methods
Cloning of HMGBI and Pf~oduction of HMGBl Mutants
The following methods were used to prepare clones and mutants of human
HMGB 1. Recombinant full length human HMGB 1 (651 base pairs; GenBank
Accession Number U51677) was cloned by PCR amplification from a human brain
Quick-Clone cDNA preparation (Clontech, Palo Alto, CA) using the following
primers; forward primer: 5' GATGGGCAAAGGAGATCCTAAG 3' (SEQ ID N0:6)
and reverse primer: 5' GCGGCCGCTTATTCATCATCATCATCTTC 3' (SEQ ID
N0:7). Human HMGB 1 mutants were cloned and purified as follows. A truncated
form of human HMGB 1 was cloned by PCR amplification from a Human Brain
Quick-Clone cDNA preparation (Clontech, Palo Alto, CA). The primers used were
(forward and reverse, respectively):
Carboxy terminus mutant (557 bp): 5' GATGGGCAAAGGAGATCCTAAG 3' (SEQ
ID N0:8) and 5' GCGGCCGC TCACTTGGTTTTTTCAGCCTTGAC 3' (SEQ ID
N0:9).
Amino terminus+B box mutant (486 bp): 5' GAGCATAAGAAGAAGCACCCA 3'
(SEQ ID NO:10) and 5' GCGGCCGC TCACTTGCTTTTTTCAGCCTTGAC 3'
(SEQ ID NO:l 1).
B box mutant (233 bp): 5' AAGTTCAAGGATCCCAATGCAAAG 3' (SEQ ID
N0:12) and 5' GCGGCCGCTCAATATGCAGCTATATCCTTTTC 3' (SEQ ID
N0:13).
Amino terminus+A box mutant (261 bp): 5' GATGGGCAAAGGAGATCCTAAG 3'
(SEQ ID NO: 14) and 5' TCACTTTTTTGTCTCCCCTTTGGG 3' (SEQ ID NO:15).
A stop codon was added to each mutant to ensure the accuracy of protein size.
PCR products were subcloned into pCRII-TOPO vector EcoRI sites using the TA
cloning method per manufacturer's instruction (Invitrogen, Carlsbad, CA).
After
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amplification, the PCR product was digested with EcoRI and subcloned into an
expression vector with a GST tag pGEX (Pharmacia); correct orientation and
positive clones were confirmed by DNA sequencing on both strands. The
recombinant plasmids were transformed into protease deficient E coli strains
BL21
or BL21(DE3)plysS (Novagen, Madison, WI) and fusion protein expression was
induced by isopropyl-D-thiogalactopyranoside (IPTG). Recombinant proteins were
obtained using affinity purification with the glutathione Sepharose resin
column
(Pharmacia).
The HMGB mutants generated as described above have the following amino
acid sequences:
Wild type HMGB 1:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKT
MSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLP SAF
FLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKE
KYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEEEDEE
DEEDEEEDDDDE (SEQ ID N0:18)
Carboxy terminus mutant:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKI3PDASVNFSEFSKKCSERWKT
MSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLP SAF
FLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKE
KYEKDIAAYRAKGKPDAAKKGVVKAEKSK (SEQ ID NO: 19)
B Box mutant: FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEM
WNNTAADDKQPYEKKAAKLKEKYEKDIAAY (SEQ ID NO: 20)
Amino terminus + A Box mutant: MGKGDPKKPTGKMSSYAFFVQTCREEHKKK
HPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPP
KGET (SEQ ID NO: 21), wherein the A box consists of the sequence
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PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK
FEDMAKADKARYEREMKTYIPPKGET (SEQ ID N0:22)
A polypeptide generated from a GST vector lacking HMGB 1 protein was
included as a control (containing a GST tag only). To inactive the bacterial
DNA
that bound to the wild type HMGB 1 and some of the mutants (carboxy terminus
and
B box), DNase I (Life Technologies), for carboxy terminus and B box mutants,
or
benzonase nuclease (Novagen, Madison, WI), for wild type HMGB1, was added at
about 20 units/ml bacteria lysate. Degradation of DNA was verified by ethidium
bromide staining of the agarose gel containing HMGB 1 proteins before and
after the
treatment. The protein eluates were passed over a polymyxin B cohunn (Pierce,
Rockford, IL) to remove any contaminating LPS, and dialyzed extensively
against
phosphate buffered saline to remove excess reduced glutathione. The
preparations
were then lyophilized and redissolved in sterile water before use. LPS levels
were
less than 60 pg/~g protein for all of the mutants and 300 pg/~g for wild type
HMG-1,
as measured by Limulus amebocyte lysate assay (Bio Whittaker Inc.,
Walkersville,
MD). The integrity of protein was verified by SDS-PAGE. Recombinant rat
HMGB 1 (Wang et al., Science 285: 248-251, 1999) was used in some experiments
since it does not have degraded fragments as observed in purified human HMGB1.
Peptide Synthesis
Peptides were synthesized and HPLC purified at Utah State University
Biotechnology Center (Logan, Utah) at 90% purity. Endotoxin was not detectable
in
the synthetic peptide preparations as measured by Limulus assay.
Cell Culture
Murine macrophage-like RAW 264.7 cells (American Type Culture
Collection, Roclcville, MD) were cultured in RPMI 1640 medium (Life
Technologies, Grand Island NY) supplemented with 10% fetal bovine serum
(Gemini, Catabasas, CA), penicillin and streptomycin (Life Technologies) and
were
used at 90% confluence in serum-free Opti-MEM I medium (Life Technologies,
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Grand Island, NY). Polymyxin B (Sigma, St. Louis, MO) was routinely added at
100-1,000 units/ml to neutralize the activity of any contaminating LPS as
previously
described; polymyxin B alone did not influence cell viability assessed with
trypan
blue (Wang et al., supra). Polymyxin B was not used in experiments of
synthetic
peptide studies.
Measuf°ement of TNF Release From Cells
TNF release was measured by a standard murine fibroblast L929 (ATCC,
American Type Culture Collection, Rockville, MD) cytotoxicity bioassay
(Bianchi et
al., Journal of Experimental Medicine 183:927-936, 1996) with the minimum
detectable concentration of 30 pg/ml. Recombinant mouse TNF was obtained from
R&D system Inc., (Minneapolis, MN). Murine fibroblast L929 cells (ATCC) were
cultured in DMEM (Life Technologies, Grand Island, NY) supplemented with 10%
fetal bovine serum (Gemini, Catabasas, CA), penicillin (50 units/ml) and
streptomycin (50 ~g/ml) (Life Technologies) in a humidified incubator with 5%
COz.
Antibody Pr~oductio~c
Polyclonal antibodies against HMGB 1 B box were raised in rabbits (Cocalico
Biologicals, Inc., Reamstown, PA) and assayed for titer by immunoblotting. IgG
was
purified from anti-HMGB 1 antiserum using Protein A agarose according to
manufacturer's instructions (Pierce, Rockford, IL). Anti-HMGB 1 B box
antibodies
were affinity purified using cyanogen bromide activated Sepharose beads
(Cocalico
Biological, Inc.). Non-immune rabbit IgG was purchased from Sigma (St. Louis,
MO). Antibodies detected full length HMGB 1 and B box in immunoassay, but did
not cross react with TNF, IL-1 and IL-6.
Labeling of HMGBI with Na-'ZSI and cell surface binding
Purified HMGB1 protein (10 ~,g) was radiolabeled with 0.2 mCi of carrier-
free lzsl (NEN Life Science Products Inc., Boston, MA) using Iodo-beads
(Pierce,
Roclcford, IL) according to the manufacturer's instructions. 'zsI_HMGB 1
protein was
separated from un-reacted'zsI by gel chromatography columns (P6 Micro Bio-Spin
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Chromatography Columns, Bio-Rad Laboratories, Hercules, CA) previously
equilibrated with 300 mM sodium chloride, 17.5 mM sodium citrate, pH 7.0, and
0.1 % bovine serum albumin (B SA). The specific activity of the eluted HMGB 1
was
about 2.8 x 106 cpm/~g protein. Cell surface binding studies were perfouned as
previously described (Yang et al., Am. J. Physiol. 275:C675-C683, 1998). RAW
264.7 cells were plated on 24-well dishes and grown to confluence. Cells were
washed twice with ice-cold PBS containing 0.1% BSA and binding was carried out
at
4°C for 2 hours with 0.5 ml binding buffer containing 120 mM sodium
chloride, 1.2
mM magnesium sulfate, 15 mM sodium acetate, 5 mM potassium chloride, 10 mM
Tris.HCl, pH 7.4, 0.2% BSA, SmM glucose and 25,000 cpm'z5I-HMGB1. At the
end of the incubation the supernatants were discarded and the cells were
washed
three times with 0.5 ml of ice-cold PBS with 0.1% BSA and lysed with 0.5 ml of
0.5
N NaOH and 0.1 % SDS for 20 minutes at room temperature. The radioactivity in
the
lysate was then measured using a gamma counter. Specific binding was
determined
as total binding minus the radioactivity obtained in the presence of an excess
amount
of unlabeled HMGB 1 or A box proteins.
Animal Expe~imev~ts
TNF knock out mice were obtained from Amgen (Thousand Oaks, CA) and
were on a B6x129 background. Age-matched wild-type B6x129 mice were used as a
control for the studies. Mice were bred in-house at the University of Florida
specific
pathogen-free transgenic mouse facility (Gainesville, FL) and were used at 6-8
weeks
of age.
Male 6-8 week old Balb/c and C3H/HeJ mice were purchased from Harlen
Sprague-Dawley (Indianapolis, IN) and were allowed to acclimate for 7 days
before
use in experiments. All animals were housed in the North Shore University
Hospital
Animal Facility under standard temperature, and a light and dark cycle.
Cecal Ligation a~cd Puncture
Cecal ligation and puncture (CLP) was performed as described previously
(Fink and Heard, J. Surg. Res. 49:186-196, 1990; Wichmann et al., Crit. Care
Med.
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26:2078-2086, 1998; and Remiclc et al., Shock 4:89-95, 1995). Briefly, Balb/c
mice
were anesthetized with 75 mg/kg ketamine (Fort Dodge, Fort Dodge, Iowa) and 20
mg/kg of xylazine (Bohringer Ingelheim, St. Joseph, MO) intramuscularly. A
midline incision was performed, and the cecum was isolated. A 6-0 prolene
suture
ligature was placed at a level 5.0 mm from the cecal tip away from the
ileocecal
valve.
The ligated cecal stump was then punctured once with a 22-gauge needle,
without direct extrusion of stool. The cecum was then placed baclc into its
normal
intra-abdominal position. The abdomen was then closed with a running suture of
6-0
prolene in two layers, peritoneum and fascia separately to prevent leakage of
fluid.
All animals were resuscitated with a normal saline solution administered sub-
cutaneously at 20 ml/kg of body weight. Each mouse received a subcutaneous
injection of imipenem (0.5 mg/mouse) (Primaxin, Merck & Co., Inc., West Point,
PA) 30 minutes after the surgery. Animals were then allowed to recuperate.
Mortality was recorded for up to 1 week after the procedure; survivors were
followed
for 2 weeks to ensure no late mortalities had occurred.
D-galactosamine Sev~sitized Mice
The D-galactosamine-sensitized model has been described previously
(Galanos et al., Proc Natl. Acad. Sci. USA 76: 5939-5943, 1979; and Lehmann et
al.,
J. Exp. Med. 165: 657-663, 1997). Mice were injected intraperitoneally with 20
mg
D-galactosamine-HCL (Sigma)/mouse (in 200.x,1 PBS) and 0.1 or 1 mg of either
HMBG1 B box or vector protein (in 200 ~,1 PBS). Mortality was recorded daily
for
up to 72 hours after injection; survivors were followed for 2 weeks, and no
later
deaths from B box toxicity were observed.
Spleen bacteria culture
Fourteen mice received either anti-HMGB 1 antibody (n=7) or control (n=7)
at 24 and 30 hours after CLP, as described herein, and were euthanized for
necropsy.
Spleen bacteria were recovered as described previously (Villa et al., J.
Endotoxin
Res. 4:197-204, 1997). Spleens were removed using sterile technique and
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homogenized in 2 ml of PBS. After serial dilutions with PBS, the homogenate
was
plated as 0.15 ml aliquots on tryptic soy agar plates (Difco, Detroit, MI) and
CFU
were counted after overnight incubation at 37°C.
Statistical Av~alysis
Data axe presented as mean ~ SEM unless otherwise stated. Differences
between groups were determined by two-tailed Student's t-test, one-way ANOVA
followed by the least significant difference test or 2 tailed Fisher's Exact
Test.
Example 2: Mapping the HMGB 1 Domains for Promotion of Cytokine Activity
HMGB 1 has 2 folded DNA binding domains (A and B boxes) and a
negatively-charged acidic caxboxyl tail. To elucidate the structural basis of
HMGB 1
cytokine activity, and to map the inflammatory protein domain, we expressed
full
length and truncated forms of HMGB 1 by mutagenesis and screened the purified
proteins for stimulating activity in monocyte cultures (FIG. 1). Full length
HMGB1,
a mutant in which the carboxy terminus was deleted, a mutant containing only
the B
box, and a mutant containing only the A box were generated. These mutants of
human HMGB 1 were made by polymerase chain reaction (PCR) using specific
primers as described herein, and the mutant proteins were expressed using a
glutathione S-transferase (GST) gene fusion system (Pharmacia Biotech,
Piscataway,
N~ in accordance with the manufacturer's instructions. Briefly, DNA fragments,
made by PCR methods, were fused to GST fusion vectors and amplified in E.
coli.
The expressed HMGB 1 protein and HMGB 1 mutants were then isolated using a GST
affinity column.
The effect of the mutants on TNF release from Murine macrophage-like
RAW 264.7 cells (ATCC) was carried out as follows. R.AW 264.7 cells were
cultured in RPMI 1640 medium (Life Technologies, Grand Island NY) supplemented
with 10% fetal bovine serum (Gemini, Catabasas, CA), penicillin and
streptomycin
(Life Technologies). Polymyxin (Sigma, St. Louis, MO) was added at 100
units/ml
to suppress the activity of any contaminating LPS. Cells were incubated with 1
~,g/ml of full length (wild-type) HMGB 1 and each HMGB 1 mutant protein in
Opti-
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MEM I medium for 8 hours. Conditioned supernatants (containing TNF which had
been released from the cells) were collected and TNF released from the cells
was
measured by a standard marine fibroblast L929 (ATCC) cytotoxicity bioassay
(Bianchi et al., supra) with the minimum detectable concentration of 30 pg/ml.
Recombinant mouse TNF was obtained from R & D Systems Inc., (Minneapolis,
MN) and used as control in these experiments. The results of this study are
shown in
FIG. 1. Data in FIG. 1 are all presented as mean + SEM unless otherwise
indicated.
(N=6-10).
As shown in FIG. 1, wild-type HMGB 1 and carboxyl-truncated HMGB 1
significantly stimulated TNF release by monocyte cultures (marine macrophage-
like
RAW 264.7 cells). The B box was a potent activator of monocyte TNF release.
This
stimulating effect of the B box was specific, because A box only weakly
activated
TNF release.
Example 3: HMGB 1 B Box Protein Promotes Cytokine Activity in a Dose
Dependent Manner
To further examine the effect of HMGB 1 B box on cytokine production,
varying amounts of HMGB1 B box were evaluated for the effects on TNF, IL-1B,
and IL-6 production in marine macrophage-like RAW 264.7 cells. RAW 264.7 cells
were stimulated with B box protein at 0-10 ~.ghnl, as indicated in FIGS. 2A-2C
for 8
hours. Conditioned media were harvested and measured for TNF, IL-1~3 and IL-6
levels. TNF levels were measured as described herein, and IL-lei and IL-6
levels
were measured using the mouse IL-lei and IL-6 enzyme-linleed immunosorbent
assay
(ELISA) kits (R&D System Inc., Minneapolis, MN) and N>5 for all experiments.
The results of the studies are shown in FIGS. 2A-2C.
As shown in FIG. 2A, TNF release from RAW 264.7 cells increased with
increased amounts of B box administered to the cells. As shown in FIG. 2B,
addition
of 1 wg/ml or 10 ~,g/ml of B box resulted in increased release of IL-1 ~3 from
RAW
264.7 cells. In addition, as shown in FIG. 2C, IL-6 release from RAW 264.7
cells
increased with increased amounts of B box administered to the cells.
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The kinetics of B box-induced TNF release were also examined. TNF release
and TNF mRNA expression were measured in RAW 264.7 cells induced by B box
polypeptide or GST tag polypeptide only used as a control (vector) (10 ~.g/ml)
for 0
to 48 hours. Supernatants were analyzed for TNF protein levels by an L929
cytotoxicity assay (N=3-5) as described herein. For mRNA measurement, cells
were
plated in 100 mm plates and treated in Opti-MEM I medium containing B box
polypeptide or the vector alone for 0, 4, 8, or 24 hours, as indicated in FIG.
2D. The
vector only sample was assayed at the 4 hour time point. Cells were scraped
off the
plate and total RNA was isolated using the RNAzoI B method in accordance with
the
manufacturer's instructions (Tel-Test "B", Inc., Friendswood, TX). TNF (287
bp)
was measured by RNase protection assay (Ambion, Austin, TX). Equal loading and
the integrity of RNA was verified by ethidium bromide staining of the RNA
sample
on an agarose-formaldehyde gel. The results of the RNase protection assay are
shown in FIG. 2D. As shown in FIG. 2D, B box activation of monocytes occurred
at
the level of gene transcription, because TNF mRNA was increased significantly
in
monocytes exposed to B box protein (FIG. 2B). TNF mRNA expression was
maximal at 4 hours and decreased at 8 and 24 hours. The vector only control
(GST
tag) showed no effect on TNF mRNA expression. A similar study was carried out
measuring TNF protein released from RAW 264.7 cells 0, 4, 8, 24, 32 or 48
hours
after administration of B box or vector only (GST tag), using the L929
cytotoxicity
assay described herein. Compared to the control (medium only), B box treatment
stimulated TNF protein expression (FIG. 2E) and vector alone (FIG. 2F) did
not.
Data are representative of three separate experiments. Together these data
indicate
that the HMGB 1 B box domain has cytolcine activity and is responsible for the
cytolcine stimulating activity of full length HMGB 1.
In summary, the HMGB 1 B box dose-dependently stimulated release of TNF,
IL-1(3 and IL-6 from monocyte cultures (FIGS. 2A-2C), in agreement with the
inflammatory activity of full length HMGB 1 (Andersson et al., J. Exp. Med.
192:
565-570, 2000). In addition, these studies indicate that maximum TNF protein
release occurred within 8 hours (FIG. 2E). This delayed pattern of TNF release
is
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similar to TNF release induced by HMGB 1 itself, and is significantly later
than the
kinetics of TNF induced by LPS (Andersson et al., supra).
Example 4: The First 20 Amino Acids of the HMGB 1 B Box Stimulate TNF Activity
The TNF-stimulating activity of the HMGB 1 B box was further mapped.
This study was carried out as follows. Fragments of the B box were generated
using
synthetic peptide protection techniques, as described herein. Five HMGB 1 B
box
fragments (from SEQ ID N0:20), containing amino acids 1-20, 16-25, 30-49, 45-
64,
or 60-74 of the HMGB 1 B box were generated, as indicated in FIG. 3. R.AW
264.7
cells were treated with B box (1 ~.g/ml) or a synthetic peptide fragment of
the B box
(10 ~,g/ml), as indicated in FIG. 3, for 10 hours and TNF release in the
supernatants
was measured as described herein. Data shown are mean ~ SEM, (n=3 experiments,
each done in duplicate and validated using 3 separate lots of synthetic
peptides). As
shown in FIG. 3, TNF-stimulating activity was retained by a synthetic peptide
corresponding to amino acids 1-20 of the HMGB1 B box of SEQ ID N0:20
(fkdpnaplcrlpsafflfcse; SEQ ID N0:23). The TNF stimulating activity of the 1-
20-
mer was less potent than either the full length synthetic B box (1-74-mer), or
full
length HMGB 1, but the stimulatory effects were specific because the synthetic
20-
mers for amino acid fragments containing 16-25, 30-49, 45-64, or 60-74 of the
HMGB 1 B box did not induce TNF release. These results are direct evidence
that
the macrophage stimulating activity of the B box specifically maps to the
first 20
amino acids of the HMGB B box domain of SEQ ID N0:20). This B box fragment
can be used in the same manner as a polypeptide encoding a full length B box
polypeptide, for example, to stimulate release of a proinflammatory cytokine,
or to
treat a condition in a patient characterized by activation of an inflammatory
cytokine
cascade.
Example 5: HMGB 1 A Box Protein Antagonizes HMGB 1 Induced Cytokine Activity
in a Dose Dependent Manner
Weale agonists are by definition antagonists. Since the HMGB 1 A box only
weakly induced TNF production, as shown in FIG. 1, the ability of HMGB 1 A box
to
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act as an antagonist of HMGB 1 activity was evaluated. This study was carried
out as
follows. Sub-confluent RAW 264.7 cells in 24-well dishes were treated with
HMGB 1 (1 wg/ml) and 0, 5, 10, or 25 ~,g/ml of A box for 16 hours in Opti-MEM
I
medium in the presence of polymyxin B (100 units/ml). The TNF-stimulating
activity (assayed using the L929 cytotoxicity assay described herein) in the
sample
receiving no A box was expressed as 100%, and the inhibition by A box was
expressed as percent of HMGB 1 alone. The results of the effect of A box on
TNF
release from RAW 264.7 cells is shown in FIG. 4A. As shown in FIG. 4A, the A
box dose-dependently inhibited HMGB 1 induced TNF release with an apparent
ECSo
of approximately 7.5 wg/ml. Data in FIG. 4A are presented as mean ~ SD (n= 2-3
independent experiments).
Example 6: HMGB 1 A Box Protein Inhibits Full Length HMGB 1 and HMGB 1 B
Box Cytolcine Activity
Antagonism of full length HMGB 1 activity by HMGB 1 A box or GST tag
(vector control) was also determined by measuring TNF release from RAW 264.7
macrophage cultures stimulated by co-addition of A box with full length HMGB
1.
RAW 264.7 macrophage cells (ATCC) were seeded into 24-well tissue culture
plates
and used at 90% confluence. The cells were treated with HMGB1, and/or A boxes
as
indicated for 16 hours in Optimum I medium (Life Technologies, Grand Island,
NY)
in the presence of polymyxin B (100 units/ml, Sigma, St. Louis, MO) and
supenlatants were collected for TNF measurement (mouse ELISA kit from R&D
System Inc, Minneapolis, MN). TNF-inducing activity was expressed as a
percentage
of the activity achieved with HMGB 1 alone. The results of these studies are
shown
in FIG. 4B. FIG. 4B is a histogram of the effect of HMGB1 (HMG-1), alone, A
box
alone, Vector (control) alone, HMGB 1 in combination with A box, and HMGB 1 in
combination with vector. As shown in FTG. 4B, HMGB 1 A box significantly
attenuated the TNF stimulating activity of full length HMGB 1.
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Example 7: HMGB1 A Box Protein Inhibits HMGB1 Cytokine Activity by Binding
to It
To determine whether the HMGB 1 A box acts as an antagonist by displacing
HMGB 1 binding, 'z5I-labeled-HMGB 1 was added to macrophage cultures and
binding was measured at 4°C after 2 hours. Binding assays in R.AW 264.7
cells were
performed as described herein. lzSl-HMGB 1 binding was measuxed in RAW 264.7
cells plated in 24-well dishes for the times indicated in FIG. SA. Specific
binding
shown equals total cell-associated lzsl-HMGB 1 (CPM/well) minus cell
associated
CPMlwell in the presence of 5,000 fold molar excess of unlabeled HMGB 1. FIG.
SA is a graph of the binding of lzsl_HMGB 1 over time. As shown in FIG. SA,
HMGB 1 exhibited saturable first order binding kinetics. The specificity of
binding
was assessed as described in Example 1.
In addition, lzsl-HMG-1 binding was measured in RAW 264.7 cells plated on
24-well dishes and incubated with lzsl HMGB 1 alone or in the presence of
unlabeled
HMGB 1 or A box. The results of this binding assay are shown in FIG. 5B. Data
represents mean ~ SEM from 3 sepaxate experiments. FIG. SB is a histogram of
the
cell surface binding of lzSl-HMGB 1 in the absence of unlabeled HMGB 1 or HMGB
1
A box, or in the presence of 5,000 molar excess of unlabeled HMGB 1 or HMGB 1
A
box, measured as a percent of the total CPM/well. In FIG. SB, "Total" equals
counts
per minutes (CPM)lwell of cell associated'z5I-HMGB1 in the absence of
unlabeled
HMGB 1 or A box for 2 hours at 4°C. "HMGB 1" or "A box" equals
CPMlwell of
cell-associated izsl_HMGB 1 in the presence of 5,000 molar excess of unlabeled
HMGB 1 or unlabeled A box. The data are expressed as the percent of total
counts
obtained in the absence of unlabeled HMGBl proteins (2,382,179 CPM/well).
These
results indicate that the HMGB 1 A box is a competitive antagonist of HMGB 1
activity ih vity~o and inhibits the TNF-stimulating activity of HMGB 1.
Example 8: Inhibition of Full Length HMGB l and HMGB 1 B Box Cytokine Activity
by Anti-B Box Polyclonal Antibodies.
The ability of antibodies directed against the HMGB 1 B box to modulated the
effect of full length or HMGB 1 B box was also assessed. Affinity purified
antibodies
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directed against the HMGB 1 B box (B box antibodies) were generated as
described
herein and using standard techniques. To assay the effect of the antibodies on
HMGB 1-induced or HMGB 1 B box-induced TNF release from RAW 264.7 cells,
sub-confluent RAW 264.7 cells in 24-well dishes were treated with HMG-1 (1
~g/ml) or HMGB1 B box (10 ~g/ml) for 10 hours with or without anti-B box
antibody (25 ~.g/ml or 100 ~.g/ml antigen affinity purified, Cocalico
Biologicals, Inc.,
Reamstovnz, PA) or non-immune IgG (25 ~,glml or 100 ~,g/ml; Sigma) added. TNF
release from the RAW 264.7 cells was measured using the L929 cytotoxicity
assay as
described herein. The results of this study are shown in FIG. 6, which is a
histogram
of TNF released by RAW 264.7 cells administered nothing, 1 ~,g/ml of HMGB1, 1
~,g/ml of HMGB 1 plus 25 ~,g/ml of anti-B box antibody, 1 ~,g/ml of HMGB 1
plus 25
~g/ml of IgG (control), 10 ~,glml of B-box, 10 ~,g/ml of B-box plus 100 ~,g/ml
of
anti-B box antibody or 10 ~,g/ml of B-box plus 100 ~.g/ml of IgG (control).
The
amount of TNF released from the cells induced by HMGB 1 alone (without
addition
of B box antibodies) was set as 100%, and the data shown in FIG. 6 are the
results of
3 independent experiments. As shown in FIG. 6, affinity purified antibodies
directed
against the HMGB 1 B box significantly inhibited TNF release induced by either
full
length HMGB 1 or the HMGB 1 B box. These results indicate that such an
antibody
can be used to modulate HMGB 1 function.
Example 9: HMGB 1 B Box Protein is Toxic to D-galactosamine-sensitized Balb/c
Mice
To investigate whether the HMGB 1 B box has cytolcine activity in vivo, we
administered HMGB 1 B box protein to unanesthetized Balb/c mice sensitized
with
D-galactosamine (D-gal), a model that is widely used to study cytokine
toxicity
(Galanos et al., supra). Briefly, mice (20-25 grams, male, Harlan Sprague-
Dawley,
Indianapolis, IN) were intraperitoneally injected with D-gal (20 mg) (Sigma,
St.
Louis, Missouri) and B box (0.1 mg/ml/mouse or 1 mg/ml/mouse) or GST tag
(vector; 0.1 mg/ml/mouse or 1 mg/ml/mouse), as indicated in Table 1. Survival
of
the mice was monitored up to 7 days to ensure no late death occurred. The
results of
this study are shown in Table 1.
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Table 1: Toxicity of HMGB 1 B box on D-galactosamine-sensitized Balb/c Mice
Treatment Alive/total
Control - 10/ 10
Vector 0.1 mg/mouse 2/2
1 mg/mouse 3/3
B box 0.1 mg/mouse 6/6
1 mg/mouse 2/8
*P<0.01 versus vector alone as tested by Fisher's Exact Test
The results of this study showed that the HMGB 1 B box was lethal to D-
galactosamine-sensitized mice in a dose-dependent mamier. In all instances in
which
death occurred, it occurred within 12 hours. Lethality was not observed in
mice
treated with comparable preparations of the purified GST vector protein devoid
of B
box.
Example 10: Histology of D-galactosainine-sensitized Balb/c Mice or C3H/HeJ
Mice
Administered HMGB 1 B Box Protein
To further assess the lethality of the HMGB 1 B box protein in vivo the
HMGB1 B box was again administered to D-galactosamine-sensitized Balb/c mice.
Mice (3 per group) received D-gal (20 mg/mouse) plus B box or vector (1
mg/mouse) intraperitorieally for 7 hours and were then sacrificed by
decapitation.
Blood was collected, and organs (liver, heart, kidney and lung) were harvested
and
fixed in 10% formaldehyde. Tissue sections were prepared with hematoxylin and
eosin staining for histological evaluation (Criterion Inc., Vancouver,
Canada). The
results of these studies axe shown in FIGS. 7A-7J, which are scanned images of
hematoxylin and eosin stained kidney sections (FIG. 7A), myocardium sections
(FIG.
7C), lung sections (FIG. 7E), and liver sections (FIGS. 7G and 7I) obtained
from an
untreated mouse and kidney sections (FIG. 7B), myocardium sections (FIG. 7D),
lung sections (FIG. 7F), and liver sections (FIGS. 7H and 7J) obtained from
mice
treated with the HMGB 1 B box. Compared to the control mice, B box treatment
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caused no abnormality in kidneys (FIGS. 7A and 7B) and lungs (FIGS. 7E and
7F).
The mice had some ischemic changes and loss of cross striation in myocardial
fibers "'
in the heart (FIGS. 7C and 7D as indicated by the arrow in FIG. 7D). Liver
showed
most of the damage by the B box as illustrated by active hepatitis (FIGS. 7G-
7J). In
FIG. 7J, hepatocyte dropouts are seen surrounded by accumulated
polymorphonuclear leukocytes. The arrows in FIG. 7J point to the sites of
polymorphonuclear accumulation (dotted) or apoptotic hepatocytes (solid).
Administration of HMGB 1 B box in vivo also stimulated significantly increased
serum levels of IL-6 (315+93 vs.20+7 pg/ml, B box vs. control, p<0.05) and IL-
1~3
(15+3 vs. 4+1 pg/ml, B box vs. control, p<0.05).
Administration of B box protein to C3H/HeJ mice (which do not respond to
endotoxin) was also lethal, indicating that HMGB 1 B box is lethal in the
absence of
LPS signal transduction. Hematoxylin and eosin stained sections of lung and
kidney
collected 8 hours after achninistration of B box revealed no abnormal
morphologic
changes. Examination of sections from the heart however, revealed evidence of
ischemia with loss of cross striation associated with amorphous pink cytoplasm
in
myocardial fibers. Sections from liver showed mild acute inflammatory
responses,
with some hepatocyte dropout and apoptosis, and occasional polymorphonuclear
leukocytes. These specific pathological changes were comparable to those
observed
after administration of full length HMGB 1 and confirm that the B box alone
can
recapitulate the lethal pathological response to HMGB 1 ivc vivo.
To address whether the TNF-stimulating activity of HMGB 1 contributes to
the mediation of lethality by B box, we measured lethality in TNF knock-out
mice
(TNF-KO, Nowak et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 278:
81202-
81209, 2000) and the wild-type controls (B6x129 strain) sensitized with D-
galactosamine (20 mg/mouse) and exposed to B box (1 mg/mouse, injected
intraperitoneally). The B box was highly lethal to the wild-type mice (6 dead
out of
nine exposed) but lethality was not observed in the TNF-KO mice treated with B
box
(0 dead out of 9 exposed, p<0.05 v. wild type). Together with the data from
the
RAW 264.7 macrophage cultures, described herein, these data now indicate that
the
B box of HMGB 1 confers specific TNF-stimulating cytokine activity.
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Example 11: HMGB 1 Protein Level is Increased in Septic Mice
To examine the role of HMGB 1 in sepsis, we established sepsis in mice and
measured serum HMGB 1 using a quantitative immunoassay described previously
(Wang et al., supra). Mice were subjected to cecal ligation and puncture
(CLP), a
well characterized model of sepsis caused by perforating a surgically-created
cecal
diverticulum, that leads to polymicrobial peritonitis and sepsis (Fink and
Heard,
supra; Wichmann et al., supra; and Remick et al., supra). Serum levels of
HMGB1
were then measured (Wang et al., supra). FIG. 8 shows the results of this
study in a
graph that illustrates the levels of HMGB 1 in mice 0 hours, 8 hours, 18
hours, 24
hours, 48 hours, and 72 hours after subjection to CLP. As shown in FIG. 8,
serum
HMGB 1 levels were not significantly increased for the first eight hours after
cecal
perforation, then increased significantly after 18 hours (FIG. 8). Increased
serum
HMGB 1 remained at elevated plateau levels for at least 72 hours after CLP, a
kinetic
profile that is quite similar to the previously-described, delayed HMGB 1
kinetics in
endotoxemia (Wang et al., supra). This temporal pattern of HMGB 1 release
corresponded closely to the development of signs of sepsis in the mice. During
the
first eight hours after cecal perforation the animals were observed to be
mildly ill,
with some diminished activity and loss of exploratory behavior. Over the
ensuing 18
hours the animals became gravely ill, huddled together in groups with
piloerection,
did not seelc water or food, and became minimally responsive to external
stimuli or
being examined by the handler.
Example 12: Treatment of Septic Mice with HMGB1 A Box Protein Increases
Survival of Mice
To determine whether the HMGB 1 A box can inhibit the lethality of HMGB 1
during sepsis, mice were subjected to cecal'perforation and treated by
administration
of A box beginning 24 hours after the onset of sepsis. CLP was performed on
male
Balb/c mice as described herein. Animals were randomly grouped, with 15-25
mice
per group. The HMGB 1 A box (60 or 600 ~,ghnouse each time) or vector (GST
tag,
600 ~,g/mouse) alone was administered intraperitoneally twice daily for 3 days
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beginning 24 hours after CLP. Survival was monitored twice daily for up to 2
weeks
to ensure no late death occurred. The results of this study are illustrated in
FIG. 9,
which is a graph of the effect of vector (GST; control) 60 ~,g/mouse or 600
~g/mouse
on survival over time (*P<0,03 vs. control as tested by Fisher's exact test).
As
shown in FIG. 9, administration of the HMGB 1 A box significantly rescued mice
from the lethal effects of sepsis, and improved survival from 28% in the
animals
treated with protein purified from the vector protein (GST) devoid of the A
box, to
68% in animals receiving A box (P<0.03 by Fischer's exact test). The rescuing
effects of the HMGB 1 A box in this sepsis model were A box dose-dependent;
animals treated with 600 ~,g/mouse of A box were observed to be significantly
more
alert, active, and to resume feeding behavior as compared to either control
animals
treated with vector-derived preparations, or to animals treated with only 60
~,g A box.
The latter animals remained gravely ill, with depressed activity and feeding
for
several days, and most died.
Example 13 : Treatment of Septic Mice with Anti-HMGB 1 Antibody Increases
Survival of Mice
Passive immunization of critically ill septic mice with anti-HMGB 1
antibodies was also assessed. In this study, male Balb/c mice (20-25 gm) were
subjected to CLP, as described herein. Affinity purified anti-HMGB1 B box
polyclonal antibody or rabbit IgG (as control) was administered at 600
~,g/mouse
beginning 24 hours after the surgery, and twice daily for 3 days. Survival was
monitored for 2 weeks. The results of this study are shown in FIG. 1 OA, which
is a
graph of the survival of septic mice treated with either a control antibody or
an anti-
HMGB 1 antibody. The results show that anti-HMGB 1 antibodies administered to
the mice 24 hours after the onset of cecal perforation significantly rescued
animals
from death as compared to administration of non-immune antibodies (p<0.02 by
Fisher's exact test). Within 12 hours after administration of anti-HMGB 1
antibodies,
treated animals showed increased activity and responsiveness as compared to
controls receiving non-immune antibodies. Whereas animals treated with non-
immune antibodies remained huddled, ill kempt, and inactive, the treated
animals
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improved significantly and within 48 hours resumed normal feeding behavior.
Anti-
HMGB 1 antibodies did not suppress bacterial proliferation in this model,
because we
observed comparable bacterial counts (CFU, the aerobic colony forming units)
from
spleen 31 hours after CLP in the treated animals as compared to animals
receiving
irrelevant antibodies (control bacteria counts = 3.50.9x104 CFU/g; n=7).
Animals
were monitored for up to 2 weeks afterwards, and late deaths were not
observed,
indicating that treatment with anti-HMGB 1 conferred complete rescue from
lethal
sepsis, and did not merely delay death.
To our lazowledge, no other specific cytokine-directed therapeutic is as
effective when administered so late after the onset of sepsis. By comparison,
administration of anti-TNF actually increases mortality in this model, and
anti-MIF
antibodies are ineffective if administered more than 8 hours after cecal
perforation
(Remick et al, supra; and Calandra et al., Nature Med. 6:164-170, 2000). These
data
demonstrate that HMGB 1 can be targeted as late as 24 hours after cecal
perforation
in order to rescue lethal cases of established sepsis.
In another example of the rescue of endotoxemic mice using anti-B box
antibodies, anti-HMGB 1 B box antibodies were evaluated for their ability to
rescue
LPS-induced septic mice. Male Balb/c mice (20-25 gm, 26 per group) were
treated
with an LD75 dose of LPS (15 mg/kg) injected intraperitoneally (IP). Anti-
HMGBl
B box or non-immune rabbit serum (0.3 ml per mouse each time, IP) was given at
time 0, +12 hours and +24 hours after LPS administration. Survival of mice was
evaluated over time. The results of this study are shown in FIG. l OB, which
is a
graph of the survival of septic mice administered anti-HMGB 1 B box antibodies
or
non-immune serum. As shown in FIG. 1 OB, anti-HMGB 1 B box antibodies
improved survival of the septic mice.
Example 14: Inhibition of HMGB 1 Signaling Pathway Using an Anti-RAGE
Antibody
Previous data implicated RAGE as an HMGB 1 receptor that can mediate
neurite outgrowth during brain development and migration of smooth muscle
cells in
wound healing (Hori et al. J. Biol. chem. 270:25752-25761, 1995; Merenmies et
al.
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J. Biol. Chem. 266:16722-16729, 1991; and Degryse et al., J. Cell Biol.
152:1197-
1206, 2001). We measured TNF release in RAW 264.7 cultures stimulated with
HMGB1 (1 ~,g/ml), LPS (0.1 ~,g/ml), or HMGB1 B box (1 ~,glml) in the presence
of
anti-RAGE antibody (25 ~,g/ml) or non-immune IgG (25 ~g/ml). Briefly, the
cells
were seeded into 24-well tissue culture plates and used at 90% confluence. LPS
(E.
coli 0111:B4, Sigma, St. Louis, MO) was sonicated for 20 minutes before use.
Cells
were treated with HMGB1 (HMG-1; 1 ~,glml), LPS (0.1 ~g/ml), or HMGB1 B box
(B Box; 1 ~,g/ml) in the presence of anti-RAGE antibody (25 ~g/ml) or non-
immune
IgG (25 ~,glml), as indicated in FIG. 11A, for 16 hours in serum-free Opti-MEM
I
medium (Life Technologies) and supernatants were collected for TNF measurement
using the L929 cytotoxicity assay described herein. IgG purified polyclonal
anti-
RAGE antibody (Catalog No. sc-8230, N-16, Santa Cruz Biotech, Inc., Santa
Cruz,
CA) was dialyzed extensively against PBS before use. The results of this study
are
shown in FIG. 11A, which is a histogram of the effects of HMGB 1, LPS, or HMGB
1
B box in the presence of anti-RAGE antibodies or non-immune IgG (control) on
TNF
release from RAW 264.7 cells. As shown in FIG. 11A, compared to non-immune
IgG, anti-RAGE antibody significantly inhibited HMGB 1 B box-induced TNF
release. This suppression was specific, because anti-RAGE did not
significantly
inhibit LPS-stimulated TNF release. Notably, the maximum inhibitory effect of
anti-
RAGE decreased HMG-1 signaling by only 40%, suggesting that other signal
transduction pathways may participate in HMGB 1 signaling.
To examine the effects of HMGB 1 or HMGB 1 B box on the
NF-xB-dependent SLAM promoter, the following experiment was carried out. RAW
264.7 macrophages were transiently co-transfected with an expression plasmid
encoding a marine MyD 88-dominant-negative (DN) mutant (corresponding to
amino acids 146-296), or empty vector, plus a luciferase reporter plasmid
under the
control of the NF-xB-dependent SLAM promoter, as described by Means et al. (J.
Immunol. 166:4074-4082, 2001). A portion of the cells were then stimulated
with
full-length HMGBl (100 ng/ml), or purified HMGB1 B box (10 ~glml), for 5
hours.
Cells were then harvested and luciferase activity was measured, using standard
methods. All transfections were performed in triplicate, repeated at least
three times,
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-65-
and a single representative experiment is shown in FIG. 11B. As shown in FIG.
118,
HMGB 1 stimulated luciferase activity in samples that were not co-transfected
with
the MyD 88 dominant negative, and the level of stimulation was decreased in
samples that were co-transfected with the MyD 88 dominant negative. This
effect
was also observed in samples administered HMGB B box.
While tlus invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by those
skilled in
the art that various changes in form and details may be made therein without
departing from the scope of the invention encompassed by the appended claims.
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SEQUENCE LISTING
<110> Critical Therapeutics, Inc.
Newman, Walter
O'Keefe, Theresa L.
<120> USE OF HMGB FRAGMENTS AS
ANTI-INFLAMMATORY AGENTS
<130> 3258.1008003
<150> 60/427,846
<151> 2002-11-20
<150> 60/427,841
<151> 2002-1l-20
<160> 58
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 215
<212> PRT
<213> Homo sapiens
<400> 1
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Tle Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 2
<211> 215
<212> PRT
<213> Mus musculus
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<400> 2
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 ~ 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 3
<211> 209
<212> PRT
<213> Homo sapiens
<400> 3
Met Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ser Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp Met Ala
50 55 60
Lys Ser Asp Lys Ala Arg Tyr Asp Arg Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Asp Lys Lys Gly Lys Lys Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu His Arg Pro Lys
100 105 110
Ile Lys Ser Glu His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Ser Glu Gln Ser Ala Lys Asp Lys Gln Pro Tyr
130 135 140
Glu Gln Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Ser Glu Ala Gly Lys Lys Gly Pro Gly
165 170 175
Arg Pro Thr Gly Ser Lys Lys Lys Asn Glu~Pro Glu Asp Glu Glu Glu
180 185 190
Glu Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu
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195 200 205
Glu
<210> 4
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 4
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser G1u
1 5 10 15
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Thr
<210> 5
<211> 69
<212> PRT
<213> Homo Sapiens
<400> 5
Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu
1 5 10 15
Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp
20 25 30
Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp
35 40 45
Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu
50 55 60
Ly~s Asp Ile Ala Ala
<210> 6
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 6
gatgggcaaa ggagatccta ag 22
<210> 7
<211> 29
<212> DNA
<213> Homo Sapiens
<400> 7
gcggccgctt attcatcatc atcatcttc 29
<210> 8
<211> 22
<212> DNA
<213> Homo Sapiens
<400> 8
gatgggcaaa ggagatccta ag 22
CA 02506328 2005-05-16
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<210>
9
<211>
32
<212>
DNA
<213> Sapiens
Homo
<400>
9
gcggccgctcacttgcttttttcagccttg ac 32
<210>
<211>
21
<212>
DNA
<213> sapiens
Homo
<400>
10
gagcataagaagaagcaccca ~ 21
<210>
11
<211>
32
<212>
DNA
<213> Sapiens
Homo
<400>
11
gcggccgctcacttgcttttttcagccttg ac 32
<210>
12
<211>
24
<212>
DNA
<213> sapiens
Homo
<400>
12
aagttcaaggatcccaatgcaaag 24
<210>
13
<211>
32
<212>
DNA
<213> Sapiens
Homo
<400>
13
gcggccgctcaatatgcagctatatccttt tc 32
<210>
14
<211>
22
<212>
DNA
<213> Sapiens
Homo
<400>
14
gatgggcaaaggagatcctaag 22
<210>
<211>
24
<212>
DNA
<213> Sapiens
Homo
<400>
15
tCaCttttttgtCtCCCCtttggg 24
<210>
16
<211>
<212>
PRT
<213> Sapiens
Homo
<400> 16
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Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu
1 5 10 15
Tyr Arg Pro Lys
<210> 17
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 17
Pro Asp Ser Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp Met
20 , 25 30
Ala Lys Ser Asp Lys Ala Arg Tyr Asp Arg Glu Met Lys Asn Tyr Val
35 40 45
Pro Pro Lys Gly Asp Lys
<210> 18
<2115 216
<212> PRT
<213> Homo Sapiens
<400> 18
Met Gly Lys Gly Asp Pro Lys Lys Pro Thr Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 ~ 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Leu Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp
195 200 205
Glu Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 19
<211> 182
<212> PRT
<213> Homo Sapiens
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<400> 19
Met Gly Lys Gly Asp Pro Lys Lys Pro Thr Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys,Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Leu Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys G1y Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys
180
<210> 20
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 20
Phe Lys Asp Pro Asn Ala Pro Lys Arg Leu Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 21
<211> 85
<212> PRT
<213> Homo Sapiens
<400> 21
Met Gly Lys Gly Asp Pro Lys Lys Pro Thr Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 ~ 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr
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<210> 22
<211> 77
<212> PRT
<213> Homo Sapiens
<400> 22
Pro Thr Gly Lys Met Ser Ser Tyr Ala Phe Phe Val Gln Thr Cys Arg
1 5 10 ~ 15
Glu Glu His Lys Lys Lys His Pro Asp Ala Ser Val Asn Phe Ser Glu
20 25 30
Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys Glu
35 40 45
Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu
50 55 60
Arg Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr
65 70 75
<210> 23
<211> 20
<212> PRT
<213> Homo Sapiens
<400> 23
Phe Lys Asp Pro Asn Ala Pro Lys Arg Leu Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu
<210> 24
<211> 216
<212> PRT
<213> Homo Sapiens
<400> 24
Met Gly Lys Gly Asp Pro Lys Lys Pro Thr Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu,Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Leu Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
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180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp
195 200 205
Glu Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 25
<211> 211
<212> PRT
<213> Homo Sapiens
<400> 25
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Ser
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Asn Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Thr His Tyr Glu Arg Gln Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr His Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln'Pro Gly
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Gln Ala Lys Gly Lys Pro Glu Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala' Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp Asp
195 200 205
Asp Asp Glu
210
<210> 26
<211> 188
<212> PRT
<213> Homo Sapiens
<400> 26
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu Cys Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Ala Met Ser Ala Lys Asp Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Val Asp Lys Asp Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Glu Asp Ser Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Leu Leu Phe Cys Ser Glu Tyr Cys Pro Lys
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100 105 110
Ile Lys Gly Glu His Pro Gly Leu Pro Ile Ser Asp Val Ala Lys Lys
115 120 125
Leu Val Glu Met Trp Asn Asn Thr Phe Ala Asp Asp Lys Gln Leu Cys
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Lys Lys Asp Thr Ala
145 150 155 160
Thr Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu
180 185
<210> 27
<211> 205
<212> PRT
<213> Homo Sapiens
<400> 27
Met Asp Lys Ala Asp Pro Lys Lys Leu Arg Gly Glu Met Leu Ser Tyr
1 5 l0 15
Ala Phe Phe Val Gln Thr Cys Gln Glu Glu His Lys Lys Lys Asn Pro
20 25 30
Asp Ala Ser Val Lys Phe Ser Glu Phe Leu Lys Lys Cys Ser Glu Thr
35 40 45
Trp Lys Thr Ile Phe Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala His Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Lys Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Leu Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Asp Asp Val Val Lys Lys
115 120 125
Leu Ala Gly Met Trp Asn Asn Thr Ala Ala Ala Asp Lys Gln Phe Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Lys Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asn Ser Ala Lys Lys Arg Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180, 185 190
Asp Glu Gln Glu Glu Glu Asn Glu Glu Asp Asp Asp Lys
195 200 205
<210> 28
<211> 80
<212> PRT
<213> Homo Sapiens
<400> 28 '
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Cys
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Trp Glu Glu His Lys Lys Gln Tyr Pro
20 25 30
Asp Ala Ser Ile Asn Phe Ser Glu Phe Ser Gln Lys Cys Pro Glu Thr
35 40 45
Trp Lys Thr Thr Ile Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Pro
50 55 60
Lys Ala Asp Lys Ala His Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
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65 70 75 80
<210> 29
<211> 80
<212> PRT
<213> Homo Sapiens
<400> 29
Lys Gln Arg Gly Lys Met Pro Ser Tyr Val Phe Cys Val Gln Thr Cys
1 5 10 15
Pro Glu Glu Arg Lys Lys Lys His Pro Asp Ala Ser Val Asn Phe Ser
20 25 30
Glu Phe Ser Lys Lys Cys Leu Val Arg Gly Lys Thr Met Ser Ala Lys
35 40 45
Glu Lys Gly Gln Phe Glu Ala Met Ala Arg Ala Asp Lys Ala Arg Tyr
50 55 60
Glu Arg Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr Lys Lys
65 70 75 80
<210> 30
<211> 86
<212> PRT
<213> Homo Sapiens
<400> 30
Met Gly Lys Arg Asp Pro Lys Gln Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Ala Gln Glu Glu His Lys Lys Lys Gln Leu
20 25 30
Asp Ala Ser Val Ser Phe Ser Glu Phe Ser Lys Asn Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Val Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Cys Tyr Glu Arg Glu Met Lys Ile Tyr Pro Tyr
65 70 75 80
Leu Lys Gly Arg Gln Lys
<210> 31
<211> 70
<212> PRT
<213> Homo Sapiens
<400> 31
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Glu Lys Met Pro Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Ala His Lys Asn Lys His Pro
20 25 30
Asp Ala Ser Val Asn Ser Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Pro Thr Lys Gln Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Arg Ala His
65 70
<210> 32
<211> 648
<212> DNA
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<213> Homo sapiens
<400> 32
atgggcaaag gagatcctaa gaagccgaca ggcaaaatgt catcatatgc attttttgtg 60
caaacttgtc gggaggagca taagaagaag cacccagatg cttcagtcaa cttctcagag 120
ttttctaaga agtgctcaga gaggtggaag accatgtctg ctaaagagaa aggaaaattt 180
gaagatatgg caaaggcgga caaggcccgt tatgaaagag aaatgaaaac ctatatccct 240
cccaaagggg agacaaaaaa gaagttcaag gatcccaatg cacccaagag gcttccttcg 300
gCCttCttCC tcttctgctc tgagtatcgc ccaaaaatca aaggagaaca tcctggcctg 360
tccattggtg atgttgcgaa gaaactggga gagatgtgga ataacactgc tgcagatgac 420
aagcagcctt atgaaaagaa ggctgcgaag ctgaaggaaa aatacgaaaa ggatatagct 480
gcatatcgag ctaaaggaaa gcctgatgca gcaaaaaagg gagttgtcaa ggctgaaaaa 540
agcaagaaaa agaaggaaga ggaggaagat gaggaagatg aagaggatga ggaggaggag 600
gaagatgaag aagatgaaga agatgaagaa gaagatgatg atgatgaa 648
<210> 33
<211> 633
<212> DNA
<213> Homo Sapiens
<400> 33
atgggcaaag gagatcctaa gaagccgaga ggcaaaatgt catcatatgc attttttgtg 60
caaacttgtc gggaggagca taagaagaag cactcagatg cttcagtcaa cttctcagag 120
ttttctaaca agtgctcaga gaggtggaag accatgtctg ctaaagagaa aggaaaattt 180
gaggatatgg caaaggcgga caagacccat tatgaaagac aaatgaaaac ctatatccct 240
cccaaagggg agacaaaaaa gaagttcaag gatcccaatg cacccaagag gcctccttcg 300
gccttcttcc tgttctgctc tgagtatcac ccaaaaatca aaggagaaca tcctggcctg 360
tccattggtg atgttgcgaa gaaactggga gagatgtgga ataacactgc tgcagatgac 420
aagcagcctg gtgaaaagaa ggctgcgaag ctgaaggaaa aatacgaaaa ggatattgct 480
gcatatcaag ctaaaggaaa gcctgaggca gcaaaaaagg gagttgtcaa agctgaaaaa 540
agcaagaaaa agaaggaaga ggaggaagat gaggaagatg aagaggatga ggaggaggaa 600
gatgaagaag atgaagaaga tgatgatgat gaa 633
<210> 34
<211> 564
<212> DNA
<213> Homo Sapiens
<400> 34
atgggcaaag gagaccctaa gaagccgaga ggcaaaatgt catcatatgc attttttgtg 60
caaacttgtc gggaggagtg taagaagaag cacccagatg cttcagtcaa cttctcagag 120
ttttctaaga agtgctcaga gaggtggaag gccatgtctg ctaaagataa aggaaaattt 180
gaagatatgg caaaggtgga caaagaccgt tatgaaagag aaatgaaaac ctatatccct 240
cctaaagggg agacaaaaaa gaagttcgag gattccaatg cacccaagag gcctccttcg 300
gcctttttgc tgttctgctc tgagtattgc ccaaaaatca aaggagagca tcctggcctg 360
cctattagcg atgttgcaaa gaaactggta gagatgtgga ataacacttt tgcagatgac 420
aagcagcttt gtgaaaagaa ggctgcaaag ctgaaggaaa aatacaaaaa ggatacagct 480
acatatcgag ctaaaggaaa gcctgatgca gcaaaaaagg gagttgtcaa ggctgaaaaa 540
agcaagaaaa agaaggaaga ggag 564
<210> 35
<211> 615
<212> DNA
<213> Homo Sapiens
<400> 35
atggacaaag cagatcctaa gaagctgaga ggtgaaatgt tatcatatgc attttttgtg 60
caaacttgtc aggaggagca taagaagaag aacccagatg cttcagtcaa gttctcagag 120
tttttaaaga agtgctcaga gacatggaag accatttttg ctaaagagaa aggaaaattt 180
gaagatatgg caaaggcgga caaggcccat tatgaaagag aaatgaaaac ctatatccct 240
cctaaagggg agaaaaaaaa gaagttcaag gatcccaatg cacccaagag gcctcctttg 300
gcctttttcc tgttctgctc tgagtatcgc ccaaaaatca aaggagaaca tcctggcctg 360
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tccattgatg atgttgtgaa gaaactggca gggatgtgga ataacaccgc tgcagctgac 420
aagcagtttt atgaaaagaa ggctgcaaag ctgaaggaaa aatacaaaaa ggatattgct 480
gcatatcgag ctaaaggaaa gcctaattca gcaaaaaaga gagttgtcaa ggctgaaaaa 540
agcaagaaaa agaaggaaga ggaagaagat gaagaggatg aacaagagga ggaaaatgaa 600
gaagatgatg ataaa 615
<210> 36
<211> 240
<212 > DNA
<213> Homo Sapiens
<400> 36
atgggcaaag gagatcctaa gaagccgaga ggcaaaatgt catcatgtgc attttttgtg 60
caaacttgtt gggaggagca taagaagcag tacccagatg cttcaatcaa cttctcagag 120
ttttctcaga agtgcccaga gacgtggaag accacgattg ctaaagagaa aggaaaattt 180
gaagatatgc caaaggcaga caaggcccat tatgaaagag aaatgaaaac ctatataccc 240
<210> 37
<211> 240
<212> DNA
<213> Homo Sapiens
<400> 37
aaacagagag gcaaaatgcc atcgtatgta ttttgtgtgc aaacttgtcc ggaggagcgt 60
aagaagaaac acccagatgc ttcagtcaac ttctcagagt tttctaagaa gtgcttagtg 120
agggggaaga ccatgtctgc taaagagaaa ggacaatttg aagctatggc aagggcagac 180
aaggcccgtt acgaaagaga aatgaaaaca tatatccctc ctaaagggga gacaaaaaaa 240
<210> 38
<211> 258
<212> DNA
<213> Homo Sapiens
<400> 38
atgggcaaaa gagaccctaa gcagccaaga ggcaaaatgt catcatatgc attttttgtg 60
caaactgctc aggaggagca caagaagaaa caactagatg cttcagtcag tttctcagag 120
ttttctaaga actgctcaga gaggtggaag accatgtctg ttaaagagaa aggaaaattt 180
gaagacatgg caaaggcaga caaggcctgt tatgaaagag aaatgaaaat atatccctac 240
ttaaagggga gacaaaaa 258
<210> 39
<211> 211
<212> DNA
<213> Homo Sapiens
<400> 39
atgggcaaag gagaccctaa gaagccaaga gagaaaatgc catcatatgc attttttgtg 60
caaacttgta gggaggcaca taagaacaaa catccagatg cttcagtcaa ctcctcagag 120
ttttctaaga agtgctcaga gaggtggaag accatgccta ctaaacagaa aggaaaattc 180
gaagatatgg caaaggcaga cagggcccat a 211
<210> 40
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 40
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
CA 02506328 2005-05-16
WO 2004/046345 PCT/US2003/037507
13/18
20 25 30
Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 . 40 45
Pro Pro Lys Gly Glu Thr
<210> 41
<211> 53
<212> PRT
<213> Homo sapiens
<400> 41
Asp Ser Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg
1 5 10 15
Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp Met Ala
20 25 30
Lys Ser Asp Lys Ala Arg Tyr Asp Arg Glu Met Lys Asn Tyr Val Pro
35 40 45
Pro Lys Gly Asp Lys
<210> 42
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 42
Pro Glu Val Pro Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Val Ser Gly Lys Glu Lys Ser Lys Phe Asp Glu Met
20 25 30
Ala Lys Ala Asp Lys Val Arg Tyr Asp Arg Glu Met Lys Asp Tyr Gly
35 40 45
Pro Ala Lys Gly Gly Lys
<210> 43
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 43
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Thr
<210> 44
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 44
Ser Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Asn Lys Cys Ser Glu
CA 02506328 2005-05-16
WO 2004/046345 PCT/US2003/037507
14/18
1 5 10 15
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Thr His Tyr Glu Arg Gln Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Thr
<210> 45
<211> 54
<212> PRT
<213> Homo sapiens
<400> 45
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Ala Met Ser Ala Lys Asp Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Val Asp Lys Ala Asp Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 40 45 i
Pro Pro Lys Gly Glu Thr
<210> 46
<21l> 54
<212> PRT
<213> Homo sapiens
<400> 46
Pro Asp Ala Ser Val Lys Phe Ser Glu Phe Leu Lys Lys Cys Ser Glu
1 5 10 15
Thr Trp Lys Thr Ile Phe Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Ala His Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Lys
<210> 47
<211> 54
<212> PRT
<213> Homo sapiens
<400> 47
Pro Asp Ala Ser Ile Asn Phe Ser Glu Phe Ser Gln Lys Cys Pro Glu
1 5 10 15
Thr Trp Lys Thr Thr Ile Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Ala His Tyr Glu Arg Glu Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Thr
<210> 48
<211> 38
<212> PRT
<213> Homo sapiens
CA 02506328 2005-05-16
WO 2004/046345 PCT/US2003/037507
15/18
<400> 48
Pro Asp Ala Ser Val Asn Ser Ser Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Pro Thr Lys Gln Gly Lys Phe Glu Asp Met Ala
20 25 30
Lys Ala Asp Arg Ala His
<210> 49
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 49
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Leu Val
1 5 10 15
Arg Gly Lys Thr Met Ser Ala Lys Glu Lys Gly Gln Phe Glu Ala Met
20 25 30
Ala Arg Ala Asp Lys Ala Arg Tyr G1u Arg Glu Met Lys Thr Tyr Ile
35 40 45
Pro Pro Lys Gly Glu Thr
<210> 50
<211> 54
<212> PRT
<213> Homo Sapiens
<400> 50
Leu Asp Ala Ser Val Ser Phe Ser Glu Phe Ser Asn Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Ser Val Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Ala Cys Tyr Glu Arg Glu Met Lys Ile Tyr Pro
35 40 45
Tyr Leu Lys Gly Arg Gln
<210> 51
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 51
Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 52
<211> 74
<212> PRT
CA 02506328 2005-05-16
WO 2004/046345 PCT/US2003/037507
16/18
<213> Homo Sapiens
<400> 52
Lys Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu His Arg Pro Lys Ile Lys Ser Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Thr Ala Lys Lys Leu Gly Glu Met Trp Ser Glu Gln
35 40 45
Ser Ala Lys Asp Lys Gln Pro Tyr Glu Gln Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 53
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 53
Phe Lys Asp Pro Asn Ala Pro Lys Arg Leu Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 54
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 54
Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr His Pro,Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Gly Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 55
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 55
Phe Lys Asp Ser Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Leu Leu
1 5 10 15
Phe Cys Ser Glu Tyr Cys Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Pro Ile Ser Asp Val Ala Lys Lys Leu Val Glu Met Trp Asn Asn Thr
CA 02506328 2005-05-16
WO 2004/046345 PCT/US2003/037507
17/18
35 40 45
Phe Ala Asp Asp Lys Gln Leu Cys Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Lys Lys Asp Thr Ala Thr Tyr
65 70
<210> 56
<211> 74
<212> PRT
<213> Homo Sapiens
<400> 56
Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Val Lys Lys Leu Ala Gly Met Trp Asn Asn Thr
35 40 45
Ala Ala Ala Asp Lys Gln Phe Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
Glu Lys Tyr Lys Lys Asp Ile Ala Ala Tyr
65 70
<210> 57
<211> 84
<212> PRT
<213> Homo Sapiens
<400> 57
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp
20 25 30
Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys
50 55 60
Ala Asp Lys A1a Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro Pro
65 70 75 80
Lys Gly Glu Thr
<210> 58
<211> 92
<212> PRT
<213> Homo Sapiens
<400> 58
Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu
1 5 10 15
Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu
20 25 30
Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr
35 40 45
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys
50 55 60
G1u Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly Lys Pro
65 70 75 80
Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys
<IMG>
