U.S. patent application number 13/736250 was filed with the patent office on 2013-05-09 for antibodies against hmgb1 and fragments thereof.
This patent application is currently assigned to THE FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH. The applicant listed for this patent is THE FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH. Invention is credited to Davorka Messmer.
Application Number | 20130115217 13/736250 |
Document ID | / |
Family ID | 37514399 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130115217 |
Kind Code |
A1 |
Messmer; Davorka |
May 9, 2013 |
ANTIBODIES AGAINST HMGB1 AND FRAGMENTS THEREOF
Abstract
In various embodiments, the present invention is drawn to
antibodies or antigen-binding fragments thereof that bind to
particular fragments of HMGB1, methods of treating a condition in a
subject characterized by activation of an inflammatory cytokine
cascade, methods of detecting and/or identifying an agent that
binds to an HMGB1 polypeptide or fragment thereof, and methods of
detecting HMGB1 in a sample.
Inventors: |
Messmer; Davorka; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH; |
Manhasset |
NY |
US |
|
|
Assignee: |
THE FEINSTEIN INSTITUTE FOR MEDICAL
RESEARCH
Manhasset
NY
|
Family ID: |
37514399 |
Appl. No.: |
13/736250 |
Filed: |
January 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11922259 |
Feb 5, 2008 |
8354106 |
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PCT/US2006/023255 |
Jun 15, 2006 |
|
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13736250 |
|
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60690983 |
Jun 16, 2005 |
|
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Current U.S.
Class: |
424/139.1 ;
435/331; 435/7.92; 436/501; 530/387.3; 530/387.9 |
Current CPC
Class: |
C07K 14/4718 20130101;
A61P 11/00 20180101; A61P 37/02 20180101; A61P 17/00 20180101; C07K
16/18 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 435/331; 435/7.92; 436/501 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. An antibody or antigen-binding fragment thereof that
specifically binds to a fragment of HMGB1, wherein said fragment of
HMGB1 is selected from the group consisting of Hp-16
(YAFFVQTCREEHKKKHPD; SEQ ID NO:5), Hp-91 (DPNAPKRPPSAFFLFCSE; SEQ
ID NO:10), Hp-106 (CSEYRPKIKGEHPGLSIG; SEQ ID NO:11) and an epitope
of HMGB1 comprising amino acid residues 106-108 of HMGB1
(Cys-Ser-Glu).
2. The antibody or antigen-binding fragment of claim 1, wherein
said antibody or antigen-binding fragment inhibits release of a
cytokine from a vertebrate cell treated with HMGB1.
3. The antibody or antigen-binding fragment of claim 2, wherein
said cytokine is selected from the group consisting of IL-6, IL-12,
TNF-.alpha., IL-18, IL-8, IL-2, IL-1.beta. and IL-5.
4. The antibody or antigen-binding fragment of claim 1, wherein
said antibody or antigen-binding fragment is an antigen-binding
fragment selected from the group consisting of an Fab fragment, an
Fab' fragment, an F(ab').sub.2 fragment and an Fv fragment.
5. The antibody or antigen-binding fragment of claim 1, wherein
said antibody or antigen-binding fragment is selected from the
group consisting of a monoclonal antibody, a human antibody, a
humanized antibody, a chimeric antibody, and an antigen-binding
fragment of any of the foregoing.
6. An isolated cell that produces the antibody or antigen-binding
fragment thereof of claim 1.
7. The isolated cell of claim 6, wherein said isolated cell is
selected from the group consisting of an immortalized B cell, a
hybridoma cell, and a recombinant cell comprising one or more
exogenous nucleic acid molecules that encode said antibody or
antigen-binding fragment thereof.
8. The isolated cell of claim 6, wherein said antibody or
antigen-binding fragment is a monoclonal antibody or an
antigen-binding fragment thereof.
9. A composition comprising the antibody or antigen-binding
fragment of claim 1 and a pharmaceutically-acceptable
excipient.
10. A method of treating a condition in a subject characterized by
activation of an inflammatory cytokine cascade comprising
administering to the subject a therapeutically effective amount of
an antibody or antigen-binding fragment thereof that specifically
binds to a fragment of HMGB1, wherein said fragment of HMGB1 is
selected from the group consisting of Hp-16 (YAFFVQTCREEHKKKHPD;
SEQ ID NO:5), Hp-31 (HPDASVNFSEFSKKCSER; SEQ ID NO:6), Hp-91
(DPNAPKRPPSAFFLFCSE; SEQ ID NO:10), Hp-106 (CSEYRPKIKGEHPGLSIG; SEQ
ID NO:11) and an epitope of HMGB1 comprising amino acid residues
106-108 of HMGB1 (Cys-Ser-Glu).
11. The method of claim 10 wherein said condition is selected from
the group consisting of sepsis, allograft rejection, arthritis,
asthma, lupus, adult respiratory distress syndrome, chronic
obstructive pulmonary disease, psoriasis, pancreatitis,
peritonitis, burns, ischemia, Behcet's disease, graft versus host
disease, inflammatory bowel disease, multiple sclerosis, and
cachexia.
12. A method of detecting and/or identifying an agent that binds to
an HMGB1 polypeptide or a fragment thereof and inhibits release of
a cytokine from a vertebrate cell treated with HMGB1, wherein said
method comprises combining: a) an antibody or antigen-binding
fragment thereof that specifically binds to a fragment of HMGB1
wherein said fragment of HMGB1 is selected from the group
consisting of Hp-16 (YAFFVQTCREEHKKKHPD; SEQ ID NO:5), Hp-31
(HPDASVNFSEFSKKCSER; SEQ ID NO:6), Hp-91 (DPNAPKRPPSAFFLFCSE; SEQ
ID NO:10), Hp-106 (CSEYRPKIKGEHPGLSIG; SEQ ID NO:11), and an
epitope of HMGB1 comprising amino acid residues 106-108 of HMGB1
(Cys-Ser-Glu); b) a test agent; and c) a composition comprising an
HMGB1 polypeptide or a fragment thereof; and detecting or measuring
the formation of a complex between said antibody or antigen-binding
fragment and said HMGB1 polypeptide or fragment thereof, wherein a
decrease in the formation of said complex relative to a suitable
control indicates that said test agent binds to said HMGB1
polypeptide or fragment thereof.
13. The antibody or antigen-binding fragment of claim 12, wherein
said cytokine is selected from the group consisting of IL-6, IL-12,
TNF-.alpha., IL-18, IL-2, IL-1.beta. and IL-5.
14. A method of detecting an HMGB1 polypeptide or a fragment
thereof in a sample comprising: a) contacting a sample with an
antibody or antigen-binding fragment thereof that specifically
binds to a fragment of HMGB1, wherein said fragment of HMGB1 is
selected from the group consisting of Hp-16 (YAFFVQTCREEHKKKHPD;
SEQ ID NO:5), Hp-31 (HPDASVNFSEFSKKCSER; SEQ ID NO:6), Hp-91
(DPNAPKRPPSAFFLFCSE; SEQ ID NO:10), Hp-106, (CSEYRPKIKGEHPGLSIG;
SEQ ID NO:11), and an epitope of HMGB1 comprising amino acid
residues 106-108 of HMGB1 (Cys-Ser-Glu) under conditions suitable
for binding of said antibody or antigen-binding fragment to said
HMGB1 polypeptide or fragment thereof present in said sample; and
b) detecting antibody-HMGB1 polypeptide complexes, antigen-binding
fragment-HMGB1 polypeptide complexes, antibody-HMGB1 fragment
complexes or antigen-binding fragment-HMGB1 fragment complexes,
wherein detection of said antibody-HMGB1 polypeptide complexes,
antigen-binding fragment-HMGB1 polypeptide complexes,
antibody-HMGB1 fragment complexes or antigen-binding fragment-HMGB1
fragment complexes is indicative of the presence of an HMGB1
polypeptide or fragment thereof in said sample.
15. The method of claim 14, wherein said antibody or
antigen-binding fragment comprises a detectable label.
16. The method of claim 14, wherein said detecting of
antibody-HMGB1 polypeptide complexes, antigen-binding
fragment-HMGB1 polypeptide complexes, antibody-HMGB1 fragment
complexes or antigen-binding fragment-HMGB1 fragment complexes is
by immunoassay.
17. The method of claim 16, wherein said immunoassay is an
ELISA.
18. A test kit for use in detecting the presence of an HMGB1
polypeptide or fragment thereof in a sample comprising: a) antibody
or antigen-binding fragment thereof of claim 1; and b) one or more
ancillary reagents suitable for detecting the presence of a complex
between said antibody or antigen-binding fragment and said HMGB1
polypeptide or fragment thereof.
Description
RELATED APPLICATION(S)
[0001] This application is a divisional of U.S. application Ser.
No. 11/922,259, which is the U.S. National Stage of International
Application No. PCT/US2006/023255, filed on Jun. 15, 2006,
published in English, which claims the benefit of U.S. Provisional
Application No. 60/690,983, filed on Jun. 16, 2005.
[0002] The entire teachings of the above applications are
incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0003] This application incorporates by reference the Sequence
Listing contained in the following ASCII text file being submitted
concurrently herewith:
[0004] a) File name: SequenceListing.txt; created: Dec. 20, 2012, 7
KB in size.
BACKGROUND OF THE INVENTION
[0005] Inflammation is often induced by proinflammatory cytokines,
such as tumor necrosis factor (TNF), interleukin (IL)-1.alpha.,
IL-1.beta., IL-6, macrophage migration inhibitory factor (MIF), and
other compounds. These proinflammatory cytokines 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 cytokine cascade.
[0006] The early proinflammatory cytokines (e.g., TNF, IL-1, etc.)
mediate inflammation, and induce the late release of high mobility
group box 1 (HMGB1; also known as HMG-1 and HMG1), a protein that
accumulates in serum and mediates delayed lethality and further
induction of early proinflammatory cytokines HMGB1 was first
identified as the founding member of a family of DNA-binding
proteins, termed high mobility group box (HMGB) proteins, which are
critical for DNA structure and stability. It was identified as a
ubiquitously-expressed nuclear protein that binds double-stranded
DNA without sequence specificity. The HMGB1 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 80 amino acid, L-shaped domains. HMG boxes are also
expressed in other transcription factors including the RNA
polymerase I transcription factor human upstream-binding factor and
lymphoid-specific factor.
[0007] HMGB1 has been implicated as a cytokine mediator of delayed
lethality in endotoxemia (Andersson, U., et al., J. Exp. Med.
192(4):565-570 (2000)). That work demonstrated that bacterial
endotoxin (lipopolysaccharide (LPS)) activates
monocytes/macrophages to release HMGB1 as a late response to
activation, resulting in elevated serum HMGB1 levels that are
toxic. Antibodies against HMGB1 prevent lethality from endotoxin
even when antibody administration is delayed until after the early
cytokine response. Like other proinflammatory cytokines, HMGB1 is a
potent activator of monocytes. It has been demonstrated that
intratracheal application of HMGB1 causes acute lung injury, and
anti-HMGB1 antibodies protect against endotoxin-induced lung edema
(Abraham, E., et al., J. Immunol. 165:2950-2954 (2000)). It has
further been shown that serum HMGB1 levels are elevated in
critically ill patients with sepsis or hemorrhagic shock, and
levels are significantly higher in non-survivors as compared to
survivors (U.S. Pat. No. 6,303,321). In vivo administration of
HMGB1 has been shown to induce arthritis when injected into murine
joints (Pullerits, R., et al., Arthritis Rheum. 481693-1700
(2003)).
[0008] HMGB1 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-.kappa.B. In addition, HMGB1 binds
Toll-like receptor 2 (TLR2) and inhibition of this interaction can
decrease or prevent inflammation (U.S. Published Application No.
20040053841). It has also been shown that receptor signal
transduction of HMGB1 occurs in part through Toll-like receptor 4
(TLR4) (Park, J. S. et al., J. Biol. Chem. 279(9):7370-77
(2004)).
[0009] The delayed kinetics of HMGB1 appearance during endotoxemia
make it a potentially good therapeutic target, but little is known
about the molecular basis of HMGB1 signaling and toxicity. Given
the importance of HMGB1 in mediating inflammation, it would be
useful to identify antibodies that bind HMGB1 and fragments
thereof, for diagnostic and therapeutic purposes.
SUMMARY OF THE INVENTION
[0010] In various embodiments, the present invention is an antibody
or antigen-binding fragment thereof that binds to a particular
fragment of HMGB1, a method of treating a condition in a subject
characterized by activation of an inflammatory cytokine cascade, a
method of detecting and/or identifying an agent that binds to a
particular fragment of HMGB1, and a method of detecting an HMGB1
polypeptide or fragment thereof in a sample.
[0011] In one embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to a
fragment of HMGB1, wherein the fragment of HMGB1 is selected from
the group consisting of Hp-16 (YAFFVQTCREEHKKKHPD; SEQ ID NO:5),
Hp-31 (HPDASVNFSEFSKKCSER; SEQ ID NO:6), Hp-91 (DPNAPKRPPSAFFLFCSE;
SEQ ID NO:10) and Hp-106 (CSEYRPKIKGEHPGLSIG; SEQ ID NO:11).
[0012] In another embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to a
fragment of HMGB1, wherein the fragment of HMGB1 is selected from
the group consisting of Hp-16 (SEQ ID NO:5), Hp-31 (SEQ ID NO:6)
and Hp-91 (SEQ ID NO:10).
[0013] In a particular embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to Hp-16
(YAFFVQTCREEHKKKHPD; SEQ ID NO:5).
[0014] In another embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to Hp-31
(HPDASVNFSEFSKKCSER; SEQ ID NO:6).
[0015] In yet another embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to Hp-91
(DPNAPKRPPSAFFLFCSE; SEQ ID NO:10).
[0016] In still another embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to Hp-106
(CSEYRPKIKGEHPGLSIG; SEQ ID NO:11).
[0017] In one embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to an
epitope of HMGB1 comprising amino acid residues 106-108 of HMGB1
(Cys-Ser-Glu).
[0018] In particular embodiments, the antibody or antigen-binding
fragment of the invention inhibits release of a cytokine (e.g.,
IL-6, IL-12, TNF-.alpha., IL-18, IL-8, IL-2, IL-1.beta. and/or
IL-5) from a vertebrate cell treated with HMGB1. In other
embodiments, the antibody or antigen-binding fragment is an
antigen-binding fragment (e.g., an Fab fragment, an Fab' fragment,
an F(ab').sub.2 fragment, an Fv fragment). In yet other
embodiments, the antibody or antigen-binding fragment is a
monoclonal antibody or an antigen-binding fragment thereof. In
still other embodiments, the antibody or antigen-binding fragment
is a human antibody, a humanized antibody, a chimeric antibody, or
an antigen-binding fragment of any of the foregoing.
[0019] In one embodiment, the invention is an isolated cell that
produces an antibody or antigen-binding fragment thereof that
specifically binds to a fragment of HMGB1, wherein said fragment of
HMGB1 is selected from the group consisting of Hp-16 (SEQ ID NO:5),
Hp-31 (SEQ ID NO:6), Hp-91 (SEQ ID NO:10) and Hp-106 (SEQ ID
NO:11).
[0020] In another embodiment, the invention is a composition
comprising an antibody or antigen-binding fragment of the invention
and a pharmaceutically-acceptable excipient.
[0021] In one embodiment, the invention is a method of treating a
condition in a subject characterized by activation of an
inflammatory cytokine cascade comprising administering to the
subject a therapeutically effective amount of an antibody or
antigen-binding fragment of the invention. In one embodiment, the
antibody or antigen-binding fragment that is administered to the
subject specifically binds to Hp-16 (SEQ ID NO:5). In another
embodiment, the antibody or antigen-binding fragment that is
administered to the subject specifically binds to Hp-31 (SEQ ID
NO:6). In yet another embodiment, the antibody or antigen-binding
fragment that is administered to the subject specifically binds to
Hp-91 (SEQ ID NO:10). In still another embodiment, the antibody or
antigen-binding fragment that is administered to the subject
specifically binds to Hp-106 (SEQ ID NO:11). In one embodiment, the
condition to be treated is sepsis, allograft rejection, arthritis,
asthma, lupus, adult respiratory distress syndrome, chronic
obstructive pulmonary disease, psoriasis, pancreatitis,
peritonitis, burns, ischemia, Behcet's disease, graft versus host
disease, inflammatory bowel disease, multiple sclerosis and/or
cachexia. In other embodiments, the condition is sepsis, arthritis
or lupus.
[0022] In one embodiment, the invention is a method of detecting
and/or identifying an agent that binds to HMGB1 or a fragment
thereof and inhibits release of a cytokine from a vertebrate cell
treated with HMGB1. In the method, an antibody or antigen-binding
fragment of the invention, a test agent, and a composition
comprising an HMGB1 polypeptide or a fragment thereof are combined,
and the formation of a complex between the antibody or
antigen-binding fragment and the HMGB1 polypeptide or fragment
thereof is detected or measured. A decrease in formation of complex
between the antibody or antigen-binding fragment and HMGB1 or
fragment thereof, as compared to a suitable control, indicates that
the test agent binds to the HMGB1 polypeptide or fragment
thereof.
[0023] In one embodiment, the invention is a method of detecting an
HMGB1 polypeptide or fragment thereof in a sample. In the method, a
sample is contacted with an antibody or antigen-binding fragment of
the invention, under conditions suitable for binding of the
antibody or antigen-binding fragment to an HMGB1 polypeptide or
fragment thereof present in the sample. If antibody-HMGB1
polypeptide complexes, antigen-binding fragment-HMGB1 polypeptide
complexes, antibody-HMGB1 fragment complexes or antigen-binding
fragment-HMGB1 fragment complexes are detected, their presence is
indicative of an HMGB1 polypeptide or fragment thereof in the
sample.
[0024] In one embodiment, the invention is a test kit for use in
detecting the presence of an HMGB1 polypeptide or fragment thereof
in a sample. In this embodiment, the test kit comprises an antibody
or antigen-binding fragment of the invention and one or more
ancillary reagents suitable for detecting the presence of a complex
between the antibody or antigen-binding fragment and an HMGB1
polypeptide or fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0026] FIG. 1A is a bar graph depicting the quantity of the
cytokine, IL-6, which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
various HMGB1-derived peptides that span the HMGB1 protein. Results
represent the mean+/-standard error of the mean (SEM) of two
independent experiments, which were conducted using DCs that were
generated from different donors.
[0027] FIG. 1B is a bar graph depicting the quantity of the
cytokine, IL-6, which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
either Hp-31, Hp-106, peptides that flank these sequences, or the
entire B-box domain (HMGB1-Bx). Untreated cells (medium) were
included as a control. Results represent the mean+/-SEM of three
independent experiments, which were conducted using DCs that were
generated from different donors.
[0028] FIG. 1C is a bar graph depicting the quantity of the
cytokine, IL-12, which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
either Hp-31, Hp-106, peptides that flank these sequences, the
entire B-box domain (HMGB1-Bx), or LPS. Untreated cells (medium)
were included as a control. Results represent the mean+/-SEM of two
independent experiments using DCs generated from different
donors.
[0029] FIG. 1D is a bar graph depicting the quantity of the
cytokine, TNF-.alpha., which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
either Hp-31, Hp-106, peptides that flank these sequences, the
entire B-box domain (HMGB1-Bx), or LPS. Untreated cells (medium)
were included as a control. Results represent the mean+/-SEM of two
independent experiments using DCs generated from different
donors.
[0030] FIG. 1E is a bar graph depicting the quantity of the
cytokine, IL-18, which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
either Hp-31, Hp-106, peptides that flank these sequences, the
entire B-box domain (HMGB1-Bx), or LPS. Untreated cells (medium)
were included as a control. Results represent the mean+/-SEM of two
independent experiments using DCs generated from different
donors.
[0031] FIG. 1F is a bar graph depicting the quantity of the
cytokine, IL-8, which is secreted by human immature
monocyte-derived dendritic cells (DCs) following stimulation with
either Hp-31, Hp-106, peptides that flank these sequences, the
entire B-box domain (HMGB1-Bx), or LPS. Untreated cells (medium)
were included as a control. Results represent the mean+/-SEM of two
independent experiments using DCs generated from different
donors.
[0032] FIG. 2A is a bar graph depicting the quantity of IL-1.beta.
that is secreted by murine bone marrow-derived dendritic cells
(BM-DCs) following stimulation with a particular HMGB1-derived
peptide (Hp-16, Hp-91 or Hp-106), the entire B-box domain
(HMGB1-Bx), or LPS. Untreated cells (medium) were included as a
control. Results represent the mean+/-SEM of three independent
experiments, which were conducted using DCs that were generated
from different donors.
[0033] FIG. 2B is a bar graph depicting the quantity of IL-12 that
is secreted by murine bone marrow-derived dendritic cells (BM-DCs)
following stimulation with a particular HMGB1-derived peptide
(Hp-16 or Hp-106), the entire B-box domain (HMGB1-Bx), or LPS.
Untreated cells (medium) were included as a control. Results
represent the mean+/-SEM of three independent experiments, which
were conducted using DCs that were generated from different
donors.
[0034] FIG. 2C is a bar graph depicting the quantity of IL-2 that
is secreted by murine bone marrow-derived dendritic cells (BM-DCs)
following stimulation with a particular HMGB1-derived peptide
(Hp-16, Hp-91 or Hp-106), the entire B-box domain (HMGB1-Bx), or
LPS. Untreated cells (medium) were included as a control. Results
represent the mean+/-SEM of three independent experiments, which
were conducted using DCs that were generated from different
donors.
[0035] FIG. 2D is a bar graph depicting the quantity of IL-8 that
is secreted by murine bone marrow-derived dendritic cells (BM-DCs)
following stimulation with a particular HMGB1-derived peptide
(Hp-16 or Hp-106), the entire B-box domain (HMGB1-Bx), or LPS.
Untreated cells (medium) were included as a control. Results
represent the mean+/-SEM of three independent experiments, which
were conducted using DCs that were generated from different
donors.
[0036] FIG. 2E is a bar graph depicting the quantity of IL-5 that
is secreted by murine bone marrow-derived dendritic cells (BM-DCs)
following stimulation with a particular HMGB1-derived peptide
(Hp-16, Hp-91 or Hp-106), the entire B-box domain (HMGB1-Bx), or
LPS. Untreated cells (medium) were included as a control. Results
represent the mean+/-SEM of three independent experiments, which
were conducted using DCs that were generated from different
donors.
[0037] FIG. 2F is a bar graph depicting the quantity of IL-18 that
is secreted by murine bone marrow-derived dendritic cells (BM-DCs)
following stimulation with a particular HMGB1-derived peptide
(Hp-16 or Hp-106), the entire B-box domain (HMGB1-Bx), or LPS.
Untreated cells (medium) were included as a control. Results
represent the mean+/-SEM of three independent experiments, which
were conducted using DCs that were generated from different
donors.
[0038] FIG. 2G is a bar graph depicting the quantity of TNF-.alpha.
that is secreted by murine bone marrow-derived dendritic cells
(BM-DCs) following stimulation with a particular HMGB1-derived
peptide (Hp-16, Hp-91 or Hp-106), the entire B-box domain
(HMGB1-Bx), or LPS. Untreated cells (medium) were included as a
control. Results represent the mean+/-SEM of three independent
experiments, which were conducted using DCs that were generated
from different donors.
[0039] FIG. 2H is a bar graph depicting the quantity of IL-12 (p70)
that is secreted by murine bone marrow-derived dendritic cells
(BM-DCs), following stimulation with a particular HMGB1-derived
peptide (Hp-31, Hp-46, non-biotinylated Hp-106 (Hp-106 (non bio))
or Hp-106). All peptides tested were biotinylated at the
N-terminus, with the exception of non-biotinylated Hp-106 (Hp-106
(non bio)). Untreated cells (medium) were included as a control.
Results represent the mean+/-SEM of three independent experiments,
which were conducted using DCs that were generated from different
donors.
[0040] FIG. 3A is a bar graph depicting expression levels of CD86
on the surface of BM-DCs following exposure to the entire B-box
domain (HMGB1-Bx), a particular HMGB1-derived peptide (Hp-16,
Hp-46, Hp-106 or Hp-121), or LPS. Untreated cells (medium) are
included as a control. One representative experiment of three
experiments is depicted.
[0041] FIG. 3B is a bar graph depicting expression levels of MHC-II
on the surface of BM-DCs following exposure to the entire B-box
domain (HMGB1-Bx), a particular HMGB1-derived peptide (Hp-16,
Hp-46, Hp-106 or Hp-121), or LPS. Untreated cells (medium) are
included as a control. One representative experiment of three
experiments is depicted.
[0042] FIG. 3C is a bar graph depicting expression levels of CD40
on the surface of BM-DCs following exposure to the entire B-box
domain (HMGB1-Bx), a particular HMGB1-derived peptide (Hp-16,
Hp-46, Hp-106 or Hp-121), or LPS. Untreated cells (medium) are
included as a control. One representative experiment of three
experiments is depicted.
[0043] FIG. 4A is a bar graph depicting .sup.3H thymidine
incorporation in allogeneic T-cells that were co-cultured with DCs,
which were isolated from C57/BL6 mice and had been exposed to
either the entire B-box domain (HMGB1-Bx) or a particular
HMGB1-derived peptide (Hp-16, Hp-46, Hp-106 or Hp-121). Responses
are reported as mean counts per minute (cpm) of thymidine
incorporation by triplicate cultures (+/-SEM). A representative
example of three independent experiments is shown as mean cpm,
+/-SEM, from triplicate cultures.
[0044] FIG. 4B is a bar graph depicting .sup.3H thymidine
incorporation in allogeneic T-cells that were co-cultured with DCs,
which were isolated from Balb/c mice and had been exposed to either
the entire B-box domain (HMGB1-Bx), LPS, or were untreated
(medium). Responses are reported as mean counts per minute (cpm) of
thymidine incorporation by triplicate cultures (+/-SEM). A
representative example of three independent experiments is shown as
mean cpm, +/-SEM, from triplicate cultures.
[0045] FIG. 5A is the amino acid sequence of a human (Homo sapiens)
HMGB1 polypeptide (SEQ ID NO:1).
[0046] FIG. 5B is an A box of a human (Homo sapiens) HMGB1
polypeptide (SEQ ID NO:2).
[0047] FIG. 5C is a B box of a human (Homo sapiens) HMGB1
polypeptide (SEQ ID NO:3).
DETAILED DESCRIPTION OF THE INVENTION
[0048] A description of example embodiments of the invention
follows.
[0049] In various embodiments, the present invention is an antibody
or antigen-binding fragment thereof that specifically binds to
HMGB1, a method of treating a condition in a subject characterized
by activation of an inflammatory cytokine cascade, a method of
detecting and/or identifying an agent that binds to HMGB1 or a
fragment thereof, and a method of detecting HMGB1 or a fragment
thereof in a sample. In particular, the invention is drawn to
antibodies or antigen-binding fragments that bind to specific
fragments of HMGB1 and methods that utilize such antibodies and
antigen-binding fragments.
Antibodies and Antibody Producing Cells
[0050] In one embodiment, the present invention encompasses
antibodies or antigen-binding fragments thereof that bind to a
fragment of HMGB1, wherein the fragment of HMGB1 is selected from
the group consisting of Hp-16 (YAFFVQTCREEHKKKHPD; SEQ ID NO:5),
Hp-31 (HPDASVNFSEFSKKCSER; SEQ ID NO:6), Hp-91 (DPNAPKRPPSAFFLFCSE;
SEQ ID NO:10) and Hp-106 (CSEYRPKIKGEHPGLSIG; SEQ ID NO:11). In a
particular embodiment, the invention is an antibody or
antigen-binding fragment thereof that specifically binds to Hp-16
(SEQ ID NO:5). In another embodiment, the invention is an antibody
or antigen-binding fragment thereof that specifically binds to
Hp-31 (SEQ ID NO:6). In yet another embodiment, the invention is an
antibody or antigen-binding fragment thereof that specifically
binds to Hp-91 (SEQ ID NO:10). In still another embodiment, the
invention is an antibody or antigen-binding fragment thereof that
specifically binds to Hp-106 (SEQ ID NO:11). As demonstrated
herein, particular fragments of HMGB1 (e.g., Hp-16, Hp-31, Hp-91
and Hp-106) induce secretion of cytokines (e.g., proinflammatory
cytokines) and chemokines. In addition, such fragments of HMGB1
also induce phenotypic and functional maturation of dendritic
cells.
[0051] The antibodies of the invention can be polyclonal or
monoclonal, and the term "antibody" is intended to encompass both
polyclonal and monoclonal antibodies. The terms polyclonal and
monoclonal refer to the degree of homogeneity of an antibody
preparation, and are not intended to be limited to particular
methods of production. In one embodiment, the antibody or
antigen-binding fragment is a monoclonal antibody or
antigen-binding fragment thereof. 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.
[0052] The term "antibody" as used herein also encompasses
functional fragments of antibodies, including fragments of
chimeric, humanized, primatized, veneered or single chain
antibodies. Functional fragments include antigen-binding fragments
of antibodies that bind to HMGB1 (e.g., a mammalian (e.g., human)
HMGB1 polypeptide). For example, antibody fragments capable of
binding to an HMGB1 polypeptide or a fragment thereof, include, but
are not limited to Fv, Fab, Fab' and F(ab').sub.2 fragments. Such
fragments can be produced by enzymatic cleavage or by recombinant
techniques. For example, papain or pepsin cleavage can generate Fab
or F(ab').sub.2 fragments, respectively. Other proteases with the
requisite substrate specificity can also be used to generate Fab or
F(ab').sub.2 fragments. Antibodies can also be produced in a
variety of truncated forms using antibody genes in which one or
more stop codons have been introduced upstream of the natural stop
site. For example, a chimeric gene encoding a F(ab').sub.2 heavy
chain portion can be designed to include DNA sequences encoding the
CH.sub.1 domain and hinge region of the heavy chain.
[0053] Single chain antibodies, and chimeric, humanized or
primatized (CDR-grafted), or veneered antibodies, as well as
chimeric, CDR-grafted or veneered single chain antibodies,
comprising portions derived from different species, and the like
are also encompassed by the present invention and the term
"antibody". The various portions of these antibodies can be joined
together chemically by conventional techniques, or can be prepared
as a contiguous protein using genetic engineering techniques. For
example, nucleic acids encoding a chimeric or humanized chain can
be expressed to produce a contiguous protein. See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,
European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO
86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.
0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and
Padlan, E. A. et al., EP 0 519 596 A1. See also, Newman, R. et al.,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody,
and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al.,
Science, 242: 423-426 (1988)) regarding single chain
antibodies.
[0054] Humanized antibodies can be produced using synthetic or
recombinant DNA technology using standard methods or other suitable
techniques. Nucleic acid (e.g., cDNA) sequences coding for
humanized variable regions can also be constructed using PCR
mutagenesis methods to alter DNA sequences encoding a human or
humanized chain, such as a DNA template from a previously humanized
variable region (see e.g., Kamman, M., et al., Nucl. Acids Res.,
17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856
(1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):
2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101:
297-302 (1991)). Using these or other suitable methods, variants
can also be readily produced. In one embodiment, cloned variable
regions can be mutated, and sequences encoding variants with the
desired specificity can be selected (e.g., from a phage library;
see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et
al., WO 93/06213).
[0055] The antibody can be a humanized antibody comprising one or
more immunoglobulin chains (e.g., an antibody comprising a CDR of
nonhuman origin (e.g., one or more CDRs derived from an antibody of
nonhuman origin) and a framework region derived from a light and/or
heavy chain of human origin (e.g., CDR-grafted antibodies with or
without framework changes)). In one embodiment, the antibody or
antigen-binding fragment thereof comprises the light chain CDRs
(CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3) of
a particular immunoglobulin. In another embodiment, the antibody or
antigen-binding fragment further comprises a human framework
region.
[0056] The antibodies and antigen-binding fragments described
herein can also be conjugated to an agent. In one embodiment, the
agent is a label, for example, a radioisotope, an epitope label
(tag), an affinity label (e.g., biotin, avidin), a spin label, an
enzyme, a fluorescent group or a chemiluminescent group. Labeled
antibodies or antigen-binding fragments of the present invention
can be used, e.g., in the diagnostic, prognostic and/or screening
methods described herein. In another embodiment, the antibody is
conjugated to a drug, toxin or anti-inflammatory agent. Conjugation
of a drug, toxin or anti-inflammatory agent to the anti-HMGB1
antibodies and antigen-binding fragments of the invention allows
for targeting of these agents to sites of HMGB1 expression and/or
activity. Drugs and toxins that can be conjugated to the antibodies
of the present invention include, for example, chemotherapeutic
agents (e.g., mitomycin C, paxlitaxol, methotrexate,
5-fluorouracil, cisplatin, cyclohexamide), toxins (e.g., ricin,
gelonin) and other agents described herein (e.g., the agents
described for combination therapy). Anti-inflammatory agents that
can be conjugated include, e.g., those described herein and known
in the art.
[0057] Antibodies that are specific for an HMGB1 polypeptide and/or
fragment thereof (e.g., a mammalian (e.g., human) HMGB1 polypeptide
and/or fragment thereof (e.g., Hp-16, Hp-31, Hp-91, Hp-106)) can be
raised against an appropriate immunogen, such as an isolated and/or
recombinant HMGB1 polypeptide or a fragment thereof (including
synthetic molecules, such as synthetic peptides). Antibodies can
also be raised by immunizing a suitable host (e.g., mouse) with
cells (e.g., GH3 pituicytes, macrophage cells (e.g., RAW 246.7
cells, human macrophage cells), peripheral blood mononuclear cells
(PBMCs (e.g., human PBMCs)), primary T cells (e.g., human primary T
cells), adrenal cells (e.g., rat adrenal PC-12 cells, human adrenal
cells), and kidney cells (e.g., rat primary kidney cells, human
primary kidney cells)) that express an HMGB1 polypeptide. In
addition, cells expressing a recombinant HMGB1 polypeptide or
fragment thereof (e.g., a mammalian (e.g., human) HMGB1 polypeptide
or fragment thereof), such as transfected cells, can be used as an
immunogen or in a screen for an antibody that binds thereto (see
e.g., Chuntharapai et al., J. Immunol., 152: 1783-1789 (1994);
Chuntharapai et al., U.S. Pat. No. 5,440,021).
[0058] Preparation of immunizing antigen, and polyclonal and
monoclonal antibody production can be performed using any suitable
technique. A variety of methods have been described (see e.g.,
Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:
511-519 (1976); Milstein et al., Nature 266: 550-552 (1977);
Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane,
1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In
Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.
M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter
11, (1991)). Generally, a hybridoma is produced by fusing a
suitable immortal cell line (e.g., a myeloma cell line such as
SP2/0, P3X63Ag8.653 or a heteromyeloma) with antibody-producing
cells. Antibody-producing cells can be obtained from the peripheral
blood or, preferably the spleen or lymph nodes, of humans or other
suitable animals immunized with the antigen of interest. The fused
cells (hybridomas) can be isolated using selective culture
conditions, and cloned by limiting dilution. Cells that produce
antibodies with the desired specificity can be selected by a
suitable assay (e.g., ELISA).
[0059] Monoclonal antibodies that bind to HMGB1 are known in the
art. For example, PCT Publication No. WO2005/026209 (the entire
teachings of which are incorporated herein by reference) describe
the production and characterization of particular monoclonal
antibodies, including "6E6 HMGB1 mAb", "2E11 HMGB1 mAb", "6H9 HMGB1
mAb", "10D4 HMGB1 mAb" and "2G7 HMGB1 mAb".
[0060] Other suitable methods of producing or isolating antibodies
of the requisite specificity (e.g., human antibodies or
antigen-binding fragments) can be used, including, for example,
methods that select recombinant antibody from a library (e.g., a
phage display library). Transgenic animals capable of producing a
repertoire of human antibodies (e.g., Xenomouse.RTM. (Abgenix,
Fremont, Calif.)) can be produced using suitable methods (see e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555
(1993); Jakobovits et al., Nature, 362: 255-258 (1993)). Additional
methods that are suitable for production of transgenic animals
capable of producing a repertoire of human antibodies have been
described (e.g., Lonberg et al., U.S. Pat. No. 5,545,806; Surani et
al., U.S. Pat. No. 5,545,807; Lonberg et al., WO97/13852).
[0061] In one embodiment, the antibody or antigen-binding fragment
thereof specifically binds to a fragment of an HMGB1 polypeptide
(e.g., a fragment of a mammalian (e.g., human) HMGB1 polypeptide).
As used herein, an antibody or antigen-binding fragment that
"specifically binds to" or "has specificity for" a fragment of
HMGB1 means that the antibody or antigen-binding fragment has an
affinity for that fragment of HMGB1 that is greater than its
affinity for other antigens. The phrases "specifically binds to"
and "has specificity for" a fragment of HMGB1 can also refer to a
binding reaction that is determinative of the presence of a target
protein (e.g., HMGB1 or a fragment thereof) in a heterogeneous
population of proteins and/or other biologics. Thus, under
designated assay conditions, the antibodies and antigen-binding
fragments of the invention bind preferentially to HMGB1 and/or
fragments of HMGB1 and do not bind in a significant amount to other
components present in a test sample.
[0062] In one embodiment, the antibody or antigen-binding fragment
thereof specifically binds to a fragment of HMGB1, wherein the
fragment of HMGB1 is selected from the group consisting of Hp-16
(SEQ ID NO:5), Hp-31 (SEQ ID NO:6), Hp-91 (SEQ ID NO:10) and Hp-106
(SEQ ID NO:11). In another embodiment, the antibody or
antigen-binding fragment thereof specifically binds to a fragment
of HMGB1, wherein the fragment of HMGB1 is selected from the group
consisting of Hp-16 (SEQ ID NO:5), Hp-31 (SEQ ID NO:6), and Hp-91
(SEQ ID NO:10). In another embodiment, the antibody or
antigen-binding fragment thereof specifically binds to Hp-16 (SEQ
ID NO:5). In yet another embodiment, the antibody or
antigen-binding fragment thereof specifically binds to Hp-31 (SEQ
ID NO:6). In still another embodiment, the antibody or
antigen-binding fragment thereof specifically binds to Hp-91 (SEQ
ID NO:10). In a further embodiment, the antibody or antigen-binding
fragment thereof specifically binds to Hp-106 (SEQ ID NO:11).
[0063] In one embodiment, the antibody or antigen-binding fragment
thereof is an IgG or an antigen-binding fragment of an IgG. In
another embodiment, the antibody or antigen-binding fragment
thereof is an IgG1 or an antigen-binding fragment of an IgG1. In
other embodiments, the antibody or antigen-binding fragment thereof
is an IgG2a, IgG2b, IgG3 antibody, or an antigen-binding fragment
of any of the foregoing.
[0064] As described and exemplified herein, particular fragments of
HMGB1 (e.g., Hp-16 (SEQ ID NO:5), Hp-31 (SEQ ID NO:6), Hp-91 (SEQ
ID NO:10) and Hp-106 (SEQ ID NO:11)) possess certain functional
properties. Accordingly, antibodies and antigen-binding fragments
that specifically bind to such HMGB1 fragments can inhibit (reduce
or prevent) one or more of functions of an HMGB1 polypeptide or
HMGB1 fragment. Such functions of HMGB1 or fragments of HMGB1
include, e.g., increasing inflammation (see, e.g., PCT Publication
No. WO 02/092004; the entire teachings of which are incorporated
herein by reference), increasing secretion or release of a cytokine
(e.g., one or more proinflammatory cytokines) from a cell (e.g., as
described herein and in PCT Publication No. WO 02/092004), binding
to RAGE, binding to TLR2, binding to TLR4, chemoattraction (see,
e.g., Degryse et al., J. Cell Biol. 152(6):1197-1206 (2001); the
entire teachings of which are incorporated herein by reference),
activation of antigen presenting cells (see, e.g., WO 03/026691;
the entire teachings of which are incorporated herein by
reference), stimulation of allogeneic T cells and induction of
phenotypic and functional maturation of dendritic cells.
[0065] In particular embodiments, the antibodies and
antigen-binding fragments of the invention inhibit release of a
cytokine from a vertebrate cell treated with HMGB1. Such cytokines,
the release of which can be inhibited by the antibodies and
antigen-binding fragments of the invention, include, e.g.,
proinflammatory cytokines and other cytokines and chemokines (e.g,
IL-6, IL-12, TNF-.alpha., IL-18, IL-8, IL-2, IL-1.beta. and/or
IL-5). In one embodiment, the antibody or antigen-binding fragment
inhibits the release of TNF-.alpha. from a vertebrate cell treated
with HMGB1. As described herein and is known in the art, an
antibody or antigen-binding fragment can be screened without undue
experimentation for the ability to inhibit release of a cytokine
(e.g., a proinflammatory cytokine) using standard methods.
[0066] In one embodiment, the antibody or antigen-binding fragment
inhibits binding of a polypeptide (e.g., RAGE, TLR2, TLR4) to
HMGB1. In another embodiment, the antibody or antigen-binding
fragment inhibits induction of phenotypic and functional maturation
of dendritic cells. In another embodiment, the antibody or
antigen-binding fragment inhibits HMGB1-mediated stimulation of
allogeneic T cells. In other embodiments, the antibodies and
antigen-binding fragments inhibit one or more functions mediated by
HMGB1 (e.g., one or more of the functions described herein).
[0067] In one embodiment, the antibody is a human antibody or an
antigen-binding fragment thereof. In another embodiment, the
antibody is a humanized antibody or an antigen-binding fragment
thereof. In another embodiment, the antibody is a chimeric antibody
or antigen-binding fragment thereof. In still another embodiment,
the antibody is a human antibody, a humanized antibody, a chimeric
antibody or an antigen-binding fragment of any of the
foregoing.
[0068] In certain embodiments, the antibodies or antigen-binding
fragments thereof specifically bind to HMGB1 epitopes or antigenic
determinants (e.g., epitopes present within HMGB1 and fragments of
HMGB1 (e.g., epitopes present within Hp-16, Hp-31, Hp-91 or
Hp-106)).
[0069] In one embodiment, the invention is a bispecific antibody,
or functional fragment thereof (e.g., F(ab').sub.2), which binds to
a fragment of HMGB1 (e.g., Hp-16, Hp-31, Hp-91 or Hp-106) and at
least one other antigen (e.g., a tumor antigen, a viral antigen).
Bispecific antibodies can be secreted by triomas and hybrid
hybridomas. Generally, triomas are formed by fusion of a hybridoma
and a lymphocyte (e.g., antibody-secreting B cell) and hybrid
hybridomas are formed by fusion of two hybridomas. Each of the
fused cells (i.e., hybridomas, lymphocytes) produces a monospecific
antibody. However, triomas and hybrid hybridomas can produce an
antibody containing antigen-binding sites that recognize different
antigens. The supernatants of triomas and hybrid hybridomas can be
assayed for bispecific antibody using a suitable assay (e.g.,
ELISA), and bispecific antibodies can be purified using
conventional methods. (See, e.g., U.S. Pat. No. 5,959,084 (Ring et
al.), U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos.
4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S.
Pat. No. 5,496,549 (Yamazaki et al.)).
[0070] In one embodiment, the invention relates to an isolated cell
that produces an antibody or an antigen-binding fragment of the
invention. In a particular embodiment, the isolated
antibody-producing cell of the invention is an immortalized cell,
such as a hybridoma, heterohybridoma, lymphoblastoid cell or a
recombinant cell. The antibody-producing cells of the present
invention have uses other than for the production of antibodies.
For example, the cell of the present invention can be fused with
other cells (such as suitably drug-marked human myeloma, mouse
myeloma, human-mouse heteromyeloma or human lymphoblastoid cells)
to produce, for example, additional hybridomas, and thus provide
for the transfer of the genes encoding the antibody. In addition,
the cell can be used as a source of nucleic acids encoding the
anti-HMGB1 immunoglobulin chains, which can be isolated and
expressed (e.g., upon transfer to other cells using any suitable
technique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567,
Winter, U.S. Pat. No. 5,225,539)). For instance, clones comprising
a sequence encoding a rearranged anti-HMGB1 light and/or heavy
chain can be isolated (e.g., by PCR). In addition, cDNA libraries
can be prepared from mRNA isolated from an appropriate cell line,
and cDNA clones encoding an anti-HMGB1 immunoglobulin chain(s) can
be isolated. Thus, nucleic acids encoding the heavy and/or light
chains of the antibodies, or portions thereof, can be obtained and
used for the production of the specific immunoglobulin,
immunoglobulin chain, or variants thereof (e.g., humanized
immunoglobulins) in a variety of host cells or in an in vitro
translation system. For example, the nucleic acids, including
cDNAs, or derivatives thereof encoding variants such as a humanized
immunoglobulin or immunoglobulin chain, can be placed into suitable
prokaryotic or eukaryotic vectors (e.g., expression vectors) and
introduced into a suitable host cell by an appropriate method
(e.g., transformation, transfection, electroporation, infection),
such that the nucleic acid is operably linked to one or more
expression control elements (e.g., in the vector or integrated into
the host cell genome), to produce a recombinant antibody-producing
cell. Thus, in certain embodiments, the invention is a nucleic acid
that encodes an antibody or antigen-binding fragment of the
invention. In other embodiments, the invention is a vector that
comprises a nucleic acid encoding an antibody or antigen-binding
fragment of the invention.
Inhibiting Release of Proinflammatory Cytokines and Methods of
Treatment
[0071] In one embodiment, the present invention is a method of
inhibiting release of a cytokine (e.g., a proinflammatory cytokine)
from a vertebrate (e.g., mammalian) cell. In one embodiment, the
method comprises treating the cell with an antibody or
antigen-binding fragment of the present invention.
[0072] As used herein, a "cytokine" is a soluble protein or peptide
that is naturally produced by mammalian cells, which regulates
immune responses and mediates cell-cell interactions. Cytokines
can, either under normal or pathological conditions, modulate the
functional activities of individual cells and tissues. A
proinflammatory cytokine is a cytokine that is capable of causing
one or more of the following physiological reactions associated
with inflammation or inflammatory conditions: vasodilation,
hyperemia, increased permeability of vessels with associated edema,
accumulation of granulocytes and mononuclear phagocytes, and
deposition of fibrin. In some cases, the proinflammatory cytokine
can also cause apoptosis. For example, in chronic heart failure, it
has been shown that TNF stimulates 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), interleukin
(IL)-1.alpha., IL-1.beta., IL-6, IL-8, IL-18, interferon .gamma.,
HMGB1, platelet-activating factor (PAF), and macrophage migration
inhibitory factor (MIF). Other cytokines, the production and/or
secretion of which can be inhibited by the antibodies and
antigen-binding fragments of the invention, include those described
and exemplified herein.
[0073] In one embodiment, the invention is a method of treating a
condition in a subject, wherein the condition is characterized by
activation of an inflammatory cytokine cascade comprising
administering to the subject an antibody or antigen-binding
fragment of the present invention.
[0074] In one embodiment, the method of treatment comprises
administering to a subject a therapeutically effective amount of an
antibody or antigen-binding fragment of the invention. As used
herein, an "effective amount" or "therapeutically effective amount"
is an amount sufficient to prevent or decrease an inflammatory
response, and/or to ameliorate and/or decrease the longevity of
symptoms associated with an inflammatory response. The amount of
antibody or antigen-binding fragment that will be effective in the
treatment, prevention or management of a particular condition can
be determined, for example, by administering the composition to an
animal model, such as those disclosed herein and/or known to those
skilled in the art. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges.
[0075] Selection of the preferred effective dose can be determined
(e.g., via clinical trials) by a skilled artisan based upon the
consideration of several factors that are known to one of ordinary
skill in the art. Such factors include, e.g., the condition or
conditions to be treated, the severity of the subject's symptoms,
the choice of antibody or antigen-binding fragment to be
administered, the subject's age, the subject's body mass, the
subject's immune status, the response of the individual subject,
and other factors known by the skilled artisan to reflect the
accuracy of administered pharmaceutical compositions.
[0076] The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of the
condition, and should be decided according to the judgment of the
practitioner and each subject's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems. For example, using an in vivo cecal
ligation and puncture (CLP) assay, a dose response assay for a
particular anti-HMGB1 monoclonal antibody, namely 6E6 HMGB1 mAb,
was performed (WO2005/026209).
[0077] For antibodies, the dosage administered to a subject (e.g.,
a human patient) is typically 0.1 mg/kg to 100 mg/kg of the
subject's body weight. Preferably, the dosage administered to a
subject is between 0.1 mg/kg and 20 mg/kg of the subject's body
weight, more preferably 1 mg/kg to 10 mg/kg of the subject's body
weight. In certain embodiments of the invention, the dosage is at
least 1 mg/kg, or at least 5 mg/kg, or at least 10 mg/kg, or at
least 50 mg/kg, or at least 100 mg/kg, or at least 150 mg/kg, of
the subject's body weight. Generally, human and humanized
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to foreign
polypeptides. Thus, lower dosages of human antibodies and less
frequent administration is often possible. For example, an
effective amount of an antibody can range from about 0.01 mg/kg to
about 5 or 10 mg/kg administered daily, weekly, biweekly or
monthly.
[0078] Methods for determining whether an antibody or
antigen-binding fragment inhibits an inflammatory condition are
known to one skilled in the art. For example, inhibition of the
release of a cytokine (e.g., a proinflammatory cytokine) from a
cell can be measured according to methods known to one skilled in
the art. For example, as described and exemplified herein,
secretion or release of particular cytokines (e.g., IL-6, IL-12,
TNF-.alpha., IL-18, IL-8, IL-2, IL-1.beta., IL-5) from dendritic
cells can be measured. In addition, TNF-.alpha. release from a cell
can be measured using 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)). The L929 cytotoxicity bioassay can be carried
out as follows. RAW 264.7 cells are cultured in RPMI 1640 medium
(Life Technologies, Grand Island, N.Y.) supplemented with 10% fetal
bovine serum (Gemini, Catabasas, Calif.), and penicillin and
streptomycin (Life Technologies). Polymyxin (Sigma, St. Louis, Mo.)
is added at 100 units/ml to suppress the activity of any
contaminating LPS. Cells are incubated with an antibody or
antigen-binding fragment of the invention in Opti-MEM I medium for
8 hours, and conditioned supernatants (containing TNF-.alpha. that
has been released from the cells) are collected. TNF-.alpha. that
is released from the cells is measured by a standard murine
fibroblast L929 (ATCC) cytotoxicity bioassay (Bianchi et al.,
supra) with the minimum detectable concentration of 30 pg/ml.
Recombinant mouse TNF-.alpha. can be obtained from R&D Systems
Inc. (Minneapolis, Minn.) and used as a control in these
experiments. Methods for measuring release of other cytokines from
cells are also known in the art.
[0079] An inflammatory condition that is suitable for the methods
of treatment described herein can be one in which the inflammatory
cytokine cascade is activated. In one embodiment, the inflammatory
cytokine cascade causes a systemic reaction, such as with endotoxic
shock. In another embodiment, the inflammatory condition is
mediated by a localized inflammatory cytokine cascade, as in
rheumatoid arthritis. Nonlimiting examples of inflammatory
conditions that can be usefully treated using the antibodies and
antigen-binding fragments of the present invention include, e.g.,
diseases involving the gastrointestinal tract and associated
tissues (such as ileus, 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 shock, immune complex disease, 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 stenosis, restenosis,
vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,
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, thryoiditis, systemic lupus erythematosus,
Goodpasture's syndrome, Behcets's syndrome, allograft rejection,
graft-versus-host disease, Type I diabetes, ankylosing 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.
[0080] In one embodiment, the condition is selected from the group
consisting of sepsis, allograft rejection, arthritis (e.g.,
rheumatoid arthritis), asthma, atherosclerosis, restenosis, 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,
ileus, multiple sclerosis, and cachexia. In another embodiment, the
condition is selected from the group consisting of sepsis,
arthritis (e.g., rheumatoid arthritis), asthma, lupus, psoriasis,
inflammatory bowel disease and Crohn's disease.
[0081] Preferably the antibodies and antigen-binding fragments are
administered to a patient in need thereof in an amount sufficient
to inhibit release of a proinflammatory cytokine from a cell and/or
to treat an inflammatory condition. 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 and/or other methods known in the art.
[0082] The terms "therapy", "therapeutic" and "treatment", as used
herein, refer to ameliorating symptoms associated with a disease or
condition, for example, an inflammatory disease or an inflammatory
condition, including preventing or delaying the onset of the
disease symptoms, and/or lessening the severity or frequency of
symptoms of the disease or condition.
[0083] The terms "subject" and "individual" are defined herein to
include animals such as mammals, including but not limited to,
primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea
pigs, rats, mice or other bovine, ovine, equine, canine, feline,
rodent, or murine species. In a preferred embodiment, the animal is
a human.
[0084] In one embodiment, an excipient can be included with the
antibodies and antigen-binding fragments of the invention. The
excipient can be selected based on the expected route of
administration of the antibodies or antigen-binding fragments 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. As described above, the dosage of the antibody or
antigen-binding fragment to be administered can be determined by
the skilled artisan without undue experimentation in conjunction
with standard dose-response studies. Depending on the condition,
the antibody or antigen-binding fragment can be administered
orally, parenterally, intranasally, vaginally, rectally, lingually,
sublingually, bucally, intrabucally and/or transdermally to the
patient.
[0085] Accordingly, antibodies or antigen-binding fragments
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 and/or
edible carrier. The antibodies or antigen-binding fragments may be
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the antibodies or
antigen-binding fragments 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.
[0086] 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
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.
[0087] The antibodies and antigen-binding fragments of the present
invention can be administered parenterally such as, for example, by
intravenous, intramuscular, intrathecal or subcutaneous injection.
Parenteral administration can be accomplished by incorporating the
antibodies and antigen-binding fragments 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, bacteriostatic saline (saline containing about 0.9% mg/ml
benzyl alcohol), phosphate-buffered saline (referred to herein as
PBS), Hank's solution, Ringer's-lactate, fixed oils, polyethylene
glycols, glycerine, propylene glycol, and other synthetic solvents.
Parenteral formulations may also include antibacterial agents
(e.g., benzyl alcohol, methyl parabens), antioxidants (e.g.,
ascorbic acid, sodium bisulfite), and chelating agents (e.g.,
EDTA). Buffers, such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride and
dextrose, may also be added. The parenteral preparation can be
enclosed in ampules, disposable syringes, or multiple dose vials
made of glass or plastic.
[0088] Rectal administration includes administering the antibodies
and antigen-binding fragments into the rectum or large intestine.
This can be accomplished using suppositories or enemas. Suppository
formulations can be made by methods known in the art. For example,
suppository formulations can be prepared by heating glycerin to
about 120.degree. C., dissolving the antibody or antigen-binding
fragment in the glycerin, mixing the heated glycerin, after which
purified water may be added, and pouring the hot mixture into a
suppository mold.
[0089] Transdermal administration includes percutaneous absorption
of the antibody or antigen-binding fragment through the skin.
Transdermal formulations include patches, ointments, creams, gels,
salves, and the like.
[0090] The antibodies and antigen-binding fragments of the present
invention can be administered nasally to a subject. As used herein,
nasally administering or nasal administration, includes
administering the antibodies or antigen-binding fragments to the
mucous membranes of the nasal passage or nasal cavity of the
subject. Pharmaceutical compositions for nasal administration of an
antibody or antigen-binding fragment include therapeutically
effective amounts of the antibody or antigen-binding fragment.
Well-known methods for nasal administration include, for example,
as a nasal spray, nasal drop, suspension, gel, ointment, cream, or
powder. Administration of the antibody or antigen-binding fragment
may also take place using a nasal tampon or nasal sponge.
[0091] As described above, a variety of routes of administration
are possible including, for example, oral, dietary, topical,
transdermal, rectal, parenteral (e.g., intravenous, intraarterial,
intramuscular, subcutaneous, intradermal injection), and inhalation
(e.g., intrabronchial, intranasal, oral inhalation, intranasal
drops). Administration can be local or systemic as indicated. The
preferred mode of administration can vary depending upon the
antibody or antigen-binding fragment to be administered and the
particular condition (e.g., disease) being treated, however, oral
or parenteral administration is generally preferred.
[0092] If desired, the antibodies or antigen-binding fragments
described herein can be administered with one or more additional
agents (e.g., agents used to treat an inflammatory condition). The
antibodies or antigen-binding fragments thereof and additional
agent(s) can be present in a single composition or administered as
separate compositions. If administered as separate compositions,
the antibodies or antigen-binding fragments thereof and additional
agent(s) can be co-administered or administered separately.
[0093] In one embodiment, the antibodies or antigen-binding
fragments of the invention are administered with an
anti-inflammatory agent. Such agents are known to one of skill in
the art. In one embodiment, the agent is 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 are IL-1.alpha., IL-1.beta., 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.beta. 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.
[0094] 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. Pat. 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); Kisich et al., J. Immunol. 163(4):2008-2016
(1999); and 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 et al., J. Immunol.
Methods 222:83-92 (1999); Lavine et al., J. Cereb. Blood Flow
Metab. 18:52-58 (1998); and Holmes et al., Hybridoma 19:363-367
(2000)). The skilled artisan can determine the amount of early
sepsis mediator to use for inhibiting any particular inflammatory
cytokine cascade without undue experimentation with routine
dose-response studies.
[0095] Other agents that can be administered with the antibodies
and antigen-binding fragments of the invention include, e.g.,
Vitaxin.TM. and other antibodies targeting .alpha.v.beta.3 integrin
(see, e.g., U.S. Pat. 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).
[0096] In one embodiment, the antibodies and antigen-binding
fragments of the invention are administered with inhibitors of TNF
biological activity (e.g., inhibitors of TNF-.alpha. biological
activity). Such inhibitors of TNF activity include, e.g., 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.RTM.; Centocor, Inc.,
Malvern, Pa.), etanercept (Enbrel.RTM.; Immunex; Seattle, Wash.),
adalimumab (Humira.RTM.; D2E7; Abbot Laboratories, Abbot Park
Ill.), CDP870 (Pharmacia Corporation; Bridgewater, N.J.) CDP571
(Celltech Group plc, United Kingdom), Lenercept (Roche,
Switzerland), and Thalidomide.
[0097] In certain embodiments, the present invention is directed to
a composition comprising the antibody or antigen-binding fragments
described herein, in a pharmaceutically-acceptable excipient. As
described above, the excipient included with the antibody or
antigen-binding fragment in these compositions is selected based on
the expected route of administration of the composition. Suitable
pharmaceutically-acceptable excipients include those described
above and known in the art.
[0098] In one embodiment, the invention is directed to an aptamer
of HMGB1 and/or a fragment of HMGB1 (e.g., an aptamer of Hp-16,
Hp-31, Hp-91 or Hp-106). As is known in the art, aptamers are
macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind
tightly to a specific molecular target (e.g., an HMGB1 polypeptide
or fragment thereof). A particular aptamer may be described by a
linear nucleotide sequence and is typically about 15-60 nucleotides
in length. The chain of nucleotides in an aptamer form
intramolecular interactions that fold the molecule into a complex
three-dimensional shape, and this three-dimensional shape allows
the aptamer to bind tightly to the surface of its target molecule.
Given the extraordinary diversity of molecular shapes that exist
within the universe of all possible nucleotide sequences, aptamers
may be obtained for a wide array of molecular targets, including
proteins and small molecules. In addition to high specificity,
aptamers have very high affinities for their targets (e.g.,
affinities in the picomolar to low nanomolar range for proteins).
Aptamers are chemically stable and can be boiled or frozen without
loss of activity. Because they are synthetic molecules, they are
amenable to a variety of modifications, which can optimize their
function for particular applications. For example, aptamers can be
modified to dramatically reduce their sensitivity to degradation by
enzymes in the blood for use in in vivo applications. In addition,
aptamers can be modified to alter their biodistribution or plasma
residence time.
[0099] Selection of apatmers that can bind HMGB1 or a fragment of
HMGB1 (e.g., Hp-16, Hp-31, Hp-91 or Hp-106) can be achieved through
methods known in the art. For example, aptamers can be selected
using the SELEX (Systematic Evolution of Ligands by Exponential
Enrichment) method (Tuerk, C., and Gold, L., Science 249:505-510
(1990)). In the SELEX method, a large library of nucleic acid
molecules (e.g., 10.sup.15 different molecules) is produced and/or
screened with the target molecule (e.g., a fragment of HMGB1). The
target molecule is allowed to incubate with the library of
nucleotide sequences for a period of time. Several methods, which
are known in the art, can then be used to physically isolate the
aptamer target molecules from the unbound molecules in the mixture,
which can be discarded. The aptamers with the highest affinity for
the target molecule can then be purified away from the target
molecule and amplified enzymatically to produce a new library of
molecules that is substantially enriched for aptamers that can bind
the target molecule. The enriched library can then be used to
initiate a new cycle of selection, partitioning, and amplification.
After 5-15 cycles of this iterative selection, partitioning and
amplification process, the library is reduced to a small number of
aptamers that bind tightly to the target molecule. Individual
molecules in the mixture can then be isolated, their nucleotide
sequences determined, and their properties with respect to binding
affinity and specificity measured and compared. Isolated aptamers
can then be further refined to eliminate any nucleotides that do
not contribute to target binding and/or aptamer structure, thereby
producing aptamers truncated to their core binding domain. See
Jayasena, S. D. Clin. Chem. 45:1628-1650 (1999) for review of
aptamer technology; the entire teachings of which are incorporated
herein by reference).
[0100] In particular embodiments, the aptamers of the invention
have the binding specificity and/or functional activity described
herein for the antibodies and antigen-binding fragments of the
invention. Thus, for example, in certain embodiments, the present
invention is drawn to aptamers that have the same or similar
binding specificity as described herein for the antibodies of the
invention (e.g., binding specificity for Hp-16, Hp-31, Hp-91 or
Hp-106). In particular embodiments, the aptamers of the invention
can bind to a fragment of HMGB1 (e.g., Hp-16, Hp-31, Hp-91 or
Hp-106) and inhibit one or more functions of the HMGB1 polypeptide.
Such HMGB1 functions include, e.g., those described herein (e.g.,
increasing inflammation, increasing secretion or release of a
cytokine (e.g., one or more proinflammatory cytokines) from a cell,
binding to RAGE, binding to TLR2, binding to TLR4, chemoattraction,
activation of antigen presenting cells, stimulation of allogeneic T
cells, and induction of phenotypic and functional maturation of
dendritic cells.
Methods of Diagnosis and/or Prognosis
[0101] In another embodiment, the invention is a diagnostic and/or
prognostic method for detecting an HMGB1 polypeptide or a fragment
thereof in a sample. In this embodiment, a sample is contacted with
an antibody or antigen-binding fragment of the present invention,
under conditions suitable for binding of the antibody or
antigen-binding fragment to HMGB1 or a fragment of HMGB1 present in
the sample. The method further comprises detecting antibody-HMGB1
complexes, antigen-binding fragment-HMGB1 complexes, antibody-HMGB1
fragment complexes or antigen-binding fragment-HMGB1 fragment
complexes, wherein detection of such complexes is indicative of the
presence of an HMGB1 polypeptide or fragment thereof in the
sample.
[0102] In another embodiment, the antibody or antigen-binding
fragment comprises a detectable label. Labels suitable for use in
detection of a complex between an HMGB1 polypeptide or fragment
thereof and an antibody or antigen-binding fragment include, for
example, a radioisotope, an epitope label (tag), an affinity label
(e.g., biotin, avidin), a spin label, an enzyme, a fluorescent
group or a chemiluminescent group.
[0103] As described herein, the antibodies and antigen-binding
fragments can be used to detect or measure expression of an HMGB1
polypeptide. For example, antibodies and antigen-binding fragments
of the present invention can be used to detect or measure an HMGB1
polypeptide in a biological sample (e.g., cells, tissues or body
fluids from an individual, such as blood, serum, leukocytes (e.g.,
activated T lymphocytes), bronchoalveolar lavage fluid, saliva,
bowel fluid, synovial fluid, biopsy specimens). In one embodiment,
the sample is blood or serum. For example, a sample (e.g., tissue
and/or fluid) can be obtained from an individual and a suitable
assay can be used to assess the presence or amount of an HMGB1
polypeptide. Suitable assays include immunological and
immunochemical methods, such as flow cytometry (e.g., FACS
analysis) and immunosorbent assays, including enzyme-linked
immunosorbent assays (ELISA), radioimmunoassay (RIA),
chemiluminescence assays, immunoblot (e.g., western blot),
immunocytochemistry and immunohistology. Generally, a sample and an
antibody or antigen-binding fragment of the present invention are
combined under conditions suitable for the formation of a complex
between an HMGB1 polypeptide and the antibody or antigen-binding
fragment thereof, and the formation of said complex is assessed
(directly or indirectly). In one embodiment, diagnosis and/or
prognosis is done using ELISA and/or western blot analysis.
[0104] As in known in the art, the presence of an increased level
of an HMGB1 polypeptide in a sample (e.g., a tissue sample)
obtained from an individual can be a diagnostic and/or prognostic
indicator for monitoring the severity and predicting the likely
clinical course of sepsis for a subject exhibiting symptoms
associated with conditions characterized by activation of the
inflammatory cascade (see U.S. Pat. No. 6,303,321, the entire
teachings of which are incorporated herein by reference). Thus, in
one embodiment, the antibodies and antigen-binding fragments of the
invention can be used in diagnostic and prognostic methods for
monitoring the severity and/or predicting the likely clinical
course of an inflammatory condition associated with HMGB1
expression (e.g., the conditions described herein). In certain
embodiments, the diagnostic and/or prognostic methods comprise
measuring the concentration of HMGB1 in a sample, preferably a
serum sample, and comparing that concentration to a standard for
HMGB1 that is representative of a normal concentration range of
HMGB1 in a like sample. In this method, a higher level of HMGB1 is
indicative of poor prognosis and/or the likelihood of toxic
reactions. The diagnostic method may also be applied to other
tissue or fluid compartments, such as cerebrospinal fluid or
urine.
[0105] In another embodiment, the invention is a test kit for use
in detecting the presence of an HMGB1 polypeptide or fragment
thereof in a sample. Such test kits can comprise, e.g., an antibody
or antigen-binding fragment of the invention and one or more
ancillary reagents suitable for detecting the presence of a complex
between the antibody or antigen-binding fragment and an HMGB1
polypeptide or fragment thereof. The antibody and antigen-binding
fragments of the present invention can be provided in lyophilized
form, either alone or in combination with additional antibodies
specific for other epitopes. The antibodies or antigen-binding
fragments thereof, which can be labeled or unlabeled, can be
included in the kits with adjunct ingredients (e.g., buffers, such
as Tris (Tris(hydroxymethyl)aminomethane), phosphate and carbonate,
stabilizers, excipients, biocides and/or inert proteins, e.g.,
bovine serum albumin). For example, the antibodies or
antigen-binding fragments can be provided as a lyophilized mixture
with the adjunct ingredients, or the adjunct ingredients can be
separately provided for combination by the user. Generally these
adjunct materials will be present in less than about 5% by weight
based on the amount of active antibody, and usually will be present
in a total amount of at least about 0.001% by weight based on
antibody concentration. Where a second antibody or antigen-binding
fragment capable of binding to the anti-HMGB1 antibody or
antigen-binding fragment is employed, such antibody or
antigen-binding fragment can be provided in the kit, for instance
in a separate vial or container. The second antibody or
antigen-binding fragment, if present, is typically labeled, and can
be formulated in an analogous manner with the antibody formulations
described above. The antibodies, antigen-binding fragments and/or
ancillary reagent of the kit can be packaged separately or together
within suitable containment means (e.g., bottle, box, envelope,
tube). When the kit comprises a plurality of individually packaged
components, the individual packages can be contained within a
single larger containment means (e.g., bottle, box, envelope,
tube).
Methods of Screening
[0106] In another embodiment, the invention is a method of
detecting and/or identifying an agent that binds to an HMGB1
polypeptide or fragment thereof and inhibits release of a cytokine
from a vertebrate cell treated with HMGB1. In this method, an
antibody or antigen-binding fragment of the invention, a test agent
and a composition comprising an HMGB1 polypeptide or fragment
thereof are combined, and complex formation between the antibody or
antigen-binding fragment and the HMGB1 polypeptide or fragment
thereof is measured. A decrease in the formation of such complex
indicates that the test agent binds to the HMGB1 polypeptide or
fragment thereof. Thus, in this embodiment, the method of detecting
or identifying an agent that binds to an HMGB1 polypeptide is a
competitive binding assay in which the ability of a test agent to
inhibit the binding of an antibody or antigen-binding fragment of
the invention is assessed. For example, in one embodiment, the
antibody or antigen-binding fragment can be labeled with a suitable
label (e.g., as described herein) and the amount of labeled
antibody or antigen-binding fragment required to saturate the HMGB1
polypeptide of fragment thereof present in the assay can be
determined. For example, a saturating amount of labeled antibody or
antigen-binding fragment and various amounts of a test agent can be
contacted with an HMGB1 polypeptide under conditions suitable for
binding, and complex formation determined. In another embodiment,
the HMGB1 polypeptide can be labeled. Suitable labels for labeling
antibodies, antigen-binding fragments and/or HMGB1 polypeptides
include those described above.
[0107] A variety of agents, such as proteins (e.g., antibodies),
peptides, peptidomimetics, small organic molecules, nucleic acids
and the like, can be tested for binding to an HMGB1 polypeptide of
fragment thereof. According to the method of the present invention,
agents can be individually screened or one or more agents can be
tested simultaneously. Where a mixture of compounds is tested, the
compounds selected by the processes described can be separated (as
appropriate) and identified using suitable methods (e.g.,
sequencing, chromatography). The presence of one or more compounds
(e.g., a ligand, inhibitor, promoter) in a test sample can also be
determined according to these methods.
[0108] Agents that bind to an HMGB1 polypeptide that are useful in
the therapeutic methods described herein can be identified, for
example, by screening libraries or collections of molecules (e.g.,
the Chemical Repository of the National Cancer Institute, in assays
described herein or using other suitable methods. Libraries, such
as combinatorial libraries, of compounds (e.g., organic compounds,
recombinant or synthetic peptides, "peptoids", nucleic acids)
produced by combinatorial chemical synthesis or other methods can
be tested (see e.g., Zuckerman, R. N. et al., J. Med. Chem., 37:
2678-2685 (1994) and references cited therein; see also, Ohlmeyer,
M. H. J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993)
and DeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913
(1993), relating to tagged compounds; Rutter, W. J. et al. U.S.
Pat. No. 5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366;
and Geysen, H. M., U.S. Pat. No. 4,833,092). Where compounds
selected from a library carry unique tags, identification of
individual compounds by chromatographic methods is possible.
[0109] The present invention will now be illustrated by the
following Examples, which is not intended to be limiting in any
way. The relevant teachings of all publications cited herein that
have not explicitly been incorporated herein by reference, are
incorporated herein by reference in their entirety.
Example 1
Four 18 Amino Acid HMGB1 Peptides Induce Cytokine Secretion in
Human and Murine Dendritic Cells
Materials and Methods:
Generation of Human Dendritic Cells (DCs)
[0110] Peripheral blood mononuclear cells (PBMCs) were isolated
from the blood of normal volunteers (Long Island Blood Services,
Melville, N.Y.) over a Ficoll-Hypaque (Amersham Biosciences,
Uppsala, Sweden) density gradient. CD14.sup.+ monocytes were
isolated from PBMCs by positive selection using anti-CD14 beads
(Miltenyi Biotech., Auburn, Calif.), following the manufacturer's
instructions. To generate DCs, CD14.sup.+ cells were cultured in
RPMI 1640 medium supplemented with 2 mM L-glutamine (GIBCO-BRL Life
Technologies, Grand Island, N.Y.), 50 .mu.M 2-mercaptoethanol
(Sigma, St. Louis, Mo.), 10 mM HEPES (GIBCO-BRL), penicillin (100
U/ml), streptomycin (100 .mu.g/ml) (GIBCO-BRL), and 5% human AB
serum (Gemini Bio-Products, Woodland, Calif.). Cultures were
maintained for 7 days in 6-well trays (3.times.10.sup.6 cells/well)
supplemented with 1000 U GM-CSF per ml (Immunex, Seattle, Wash.)
and 200 U IL-4 per ml (R&D Systems, Minneapolis, Minn.) at days
0, 2, 4 and 6.
Generation of Mouse DCs
[0111] Bone marrow-derived DCs (BM-DCs) were generated using
modifications of the original method described by Inaba, et al. (J.
Exp. Med. 176:1693-1702 (1992)). In brief, bone marrow suspensions
were incubated with red cell lysis buffer (PUREGENE.TM. RBC Lysis
Solution, Gentra Systems, Minneapolis, Minn.) to remove red blood
cells. After washing in media, lymphocytes and Ia-positive cells
were killed with a cocktail of monoclonal antibodies (mAbs) and
rabbit complement for 60 min at 37.degree. C. The mAbs that were
used were GK1.5 anti-CD4, TIB211 anti-CD8, TIB 120 anti-Ia, and TIB
146 anti B220 (these mAbs were kindly provided by Dr. Ralph
Steinman). The cells were subsequently cultured in media containing
5% FCS and 10 ng/ml recombinant mouse GM-CSF (R&D Systems,
Minneapolis, Minn.) for 7 days. For some experiments, the cells
were further purified at day 7 using CD11c.sup.+-microbeads
(Miltenyi Biotech., Auburn, Calif.), according to the
manufacturer's instructions.
Reagents
[0112] Recombinant HMGB1-B box domain (HMGB1-Bx) was expressed in
Escherichia coli and purified as described by Li, et al. (Mol. Med.
9:37-45; J. Immunol. Methods 289:211-223 (2004)). Purified HMGB1-Bx
contained trace amounts of LPS (19 pg LPS/.about.g HMGB1-Bx) as
measured by the chromogenic Limulus amebocyte lysate assay
(BioWhittacker Inc, Walkersville, Md.). All experiments using
HMGB1-Bx, as well as the HMGB1 peptides, were performed in the
presence of polymyxin B (200 U/ml) to neutralize the amount of
contaminating LPS in the HMGB1-Bx and peptide preparations. We have
previously shown that the DC stimulatory capacity of HMGB1-Bx
requires an intact tertiary structure and is not due to
contaminating amounts of LPS, as trypsinization abolished HMGB1-Bx
activity (Messmer, et al., J. Immunol. 173: 307-313 (2004); the
entire teachings of which are incorporated herein by
reference).
Treatment of Dendritic Cells and Measurements of Cytokines and
Chemokines
[0113] Secreted cytokine/chemokine levels were measured by ELISA
(Pierce Boston Technology Center, SearchLight.TM. Proteome Arrays
Multiplex Sample Testing Services, Woburn, Mass.) 48 h after
addition of the various peptides. Polymyxin B (200 U/ml) was added
to all cultures, except those containing LPS, before the stimuli
were added. Immature monocyte-derived human dendritic cells (DCs)
and immature bone-marrow derived murine dendritic cells (BM-DCs)
were cultured either in the presence of HMGB1 peptides
(200.about.g/ml), whose sequences map to different regions of the
HMGB1 protein (see Table 1), the entire HMGB1 B-box domain
(HMGB1-Bx) (50.about.g/ml), or LPS (100 ng/ml). Untreated cells
(medium) were tested as a control. Each peptide was named according
to the corresponding position of its first amino acid within the
full-length HMGB1 sequence. All peptides were N-terminally
biotinylated except "Hp-106 (non bio)".
TABLE-US-00001 TABLE 1 Amino Acid Sequences of HMGB1 Peptides
Peptide Name Amino Acid sequence Hp-1 MGKGDPKKPRGKMSSYAF (SEQ ID
NO: 4) Hp-16 YAFFVQTCREEHKKKHPD (SEQ ID NO: 5) Hp-31
HPDASVNFSEFSKKCSER (SEQ ID NO: 6) Hp-46 SERWKTMSAKEKGKFEDM (SEQ ID
NO: 7) Hp-61 EDMAKADKARYEREMKTY (SEQ ID NO: 8) Hp-76
KTYIPPKGETKKKFKDPN (SEQ ID NO: 9) Hp-91 DPNAPKRPPSAFFLFCSE (SEQ ID
NO: 10) Hp-106 CSEYRPKIKGEHPGLSIG (SEQ ID NO: 11) Hp-113
IKGEHPGLSIGDVAKKLG (SEQ ID NO: 12) Hp-121 SIGDVAKKLGEMWNNTAA (SEQ
ID NO: 13) Hp-133 WNNTAADDKQPYEKKAAK (SEQ ID NO: 14) Hp-136
TAADDKQPYEKKAAKLKE (SEQ ID NO: 15) Hp-151 LKEKYEKDIAAYRAKGKP (SEQ
ID NO: 16) Hp-166 GKPDAAKKGVVKAEKSKK (SEQ ID NO: 17) Hp-181
SKKKKEEEEDEEDEEDEE (SEQ ID NO: 18) Hp-196 DEEEEEDEEDEDEEEDDDDE (SEQ
ID NO: 19)
Results
[0114] It has been shown previously that an 18 amino acid peptide,
whose sequence corresponds to a part of the B box domain of HMGB1,
induced IL-6 secretion in human monocyte-derived DCs (Messmer, et
al., J. Immunol. 173: 307-313 (2004)). In order to identify other
peptides that induce cytokine secretion, various 18 amino acid
HMGB1 peptides that span the whole HMGB1 molecule (see Table 1)
were tested. Peptides Hp-31 and Hp-106 induced secretion of IL-6 by
DCs (FIG. 1A). Subsequently, peptides that overlap by three amino
acids with either the N- or C-terminus of these two peptides were
tested. Hp-91, a C-terminal flanking peptide of Hp-106, which
shares only three amino acids (namely CSE; see Table 1) with
Hp-106, also enhanced IL-6 secretion by DCs (FIG. 1B). In contrast,
two peptides that flank and partially overlap with Hp-31 (i.e.,
Hp-16 and Hp-46) did not induce IL-6 secretion when tested (FIG.
1B). An Hp-106 peptide that was not biotinylated at its N-terminus
also failed to stimulate IL-6 secretion, indicating that N-terminal
biotinylation is required for the DC-stimulatory effect of the
active peptide (see FIG. 1B, labeled as "Hp-106 (non bio)").
Stimulation of IL-6 secretion, however, was not caused by biotin,
because several different peptides that were N-terminally
biotinylated did not stimulate secretion of IL-6 by DCs (FIGS. 1A
and 1B). In addition, three peptides, Hp-31, Hp-91, and Hp-106,
which induced IL-6 secretion, also induced secretion of IL-12 (p
70) (FIG. 1C), TNF-.about.(FIG. 1D), and IL-18 (FIG. 1E), but did
not induce secretion of IL-8 (FIG. 1F). In contrast, HMGB1-Bx
enhanced production of IL-8, but not IL-18 (FIGS. 1E and 1F).
Neither HMGB1-Bx-treated DCs, nor the peptide-treated DCs, showed
enhanced secretion of IL-10 (Table 2) or TGF-.beta..
[0115] Murine BM-DCs that were exposed to HMGB1-Bx displayed
enhanced secretion of IL-1.beta., IL-2, IL-5, TNF-.alpha., IL-12
(p70), and IL-8 (FIGS. 2A-2E and 2G), but not IL-18 (FIG. 2F). In
contrast to human DCs, HMGB1-Bx-stimulated murine BM-DCs did not
show enhanced secretion of IL-6 (Table 2). The three HMGB1 peptides
that induced cytokine secretion by human DCs (i.e., Hp-106, Hp-91
and Hp-31), also induced cytokine secretion by murine BM-DCs. In
addition, Hp-16, which did not stimulate cytokine secretion in
human DCs, enhanced cytokine secretion in murine BM-DCs. Hp-16 and
Hp-106 enhanced secretion of IL-1.beta., IL-2, IL-5, IL-12, and
IL-18 (FIGS. 2A-2C, 2E and 2G), but only Hp-106 enhanced secretion
of IL-8 and TNF-.alpha. (FIGS. 2D and 2G). IL-18 production was
enhanced by exposure of BM-DCs to either Hp-16 or Hp-106, but not
to HMGB1-Bx (FIG. 2F). Hp-91, which enhanced cytokine secretion in
human DCs, also increased production of IL-1.beta., IL-2, and IL-5
(FIGS. 2A, 2C and 2E), but not of TNF-.alpha. (FIG. 2G), IL-18, or
IL-8 in BM-DCs.
[0116] Hp-31 enhanced the production of IL-12 (p70) (FIG. 2H),
IL-2, IL-5, and IL-1.beta., but not IL-6 and IL-10 (Table 2) in
murine BM-DCs. Furthermore, as observed for human DCs, N-terminal
biotinylation was required to stimulate cytokine secretion. The
non-biotinylated Hp-106 peptide ("Hp-106 (non-bio)") did not
enhance IL-12 secretion (FIG. 2H). The DC stimulatory capacity of
the peptides was dependent on the peptide sequence and not biotin,
because certain biotinylated peptides did not enhance IL-12
secretion (FIG. 2H).
TABLE-US-00002 TABLE 2 Profile of Cytokine Secretion in Human and
Murine Dendritic Cells Following Stimulation with HMGB1-Bx or
Select HMGB1 Peptides. HMGB1-Bx Hp-16 Hp-31 Hp-91 Hp-106 HUMAN DC
IL-6 + - + + + IL-12 + - + + + TNF.alpha. + - + + + IL-18 - - + + +
IL-8 + - - - - IL-10 - - - - - IL-2 - n.a. n.a. n.a. n.a.
IL-1.beta. - n.a. n.a. n.a. n.a. IL-5 n.a. n.a. n.a. n.a. n.a.
MURINE DC IL-6 - - - - - IL-12 + + + + + TNF.alpha. + - n.a. - +
IL-18 - + n.a. - + IL-8 + - n.a. - + IL-10 - - - - - IL-2 + + + + +
IL-1.beta. + + + + + IL-5 + + + + + "n.a." = not analyzed; "+"
indicates an increase; and "-" indicates no change relative to
untreated cells (medium). The cytokine levels (pg/ml) were measure
by ELISA 48 h after exposure to HMGB1-Bx or the particular HMGB1
peptide.
[0117] Of the 16 HMGB1 peptides that were tested, 3 peptides (i.e.,
Hp-31, Hp-91 and Hp-106) induced cytokine secretion in both human
and murine DCs, while peptide Hp-16 stimulated cytokine secretion
in murine, but not human DCs. These results indicate that peptides
Hp-31, Hp-91, Hp-106 and Hp-16 could be used to produce antibodies
having potential anti-inflammatory properties.
Example 2
Select HMGB1 Peptides Induce Phenotypic Maturation of Murine
BM-DCs
Materials and Methods
[0118] In order to determine whether HMGB1-Bx and/or particular
HMGB1 peptides could induce phenotypic maturation of murine DCs,
immature BM-DCs were exposed to either HMGB1-Bx, a particular HMGB1
peptide, or LPS (FIG. 3). Fluorescence activated cell sorting
(FACS) analysis was performed on immature DCs that were cultured in
the presence of either HMGB1-Bx (50 .mu.g/ml), an HMGB1 peptide
(200 .mu.g/ml), or LPS (100 ng/ml). Untreated DCs (medium) were
also tested as a control. DCs were gated on CD11c.sup.+ cells and
analyzed for expression of specific maturation markers (e.g., CD86,
MHC-II, CD40) by surface membrane immunofluorescence. In
particular, 1.times.10.sup.4 DCs were reacted for at least 20 min
at 4.degree. C. in 100 ml of PBS/5% FCS/0.1% sodium azide (staining
buffer) with fluorescein isothiocyanate (FITC)-conjugated IgG
monoclonal antibodies (mAbs) that are specific for CD86, CD40 or
MHC-II (eBioscience). Cells were then washed 4 times with staining
buffer, fixed in 10% formaldehyde in PBS (pH 7.2-7.4) and examined
by flow cytometry using a FACScan (BD Biosciences). In all
experiments, isotype controls were included using a FITC-conjugated
irrelevant mAb of the same Ig class. Results are depicted as mean
fluorescence intensity (MFI).
Results
[0119] HMGB1-Bx induced a small increase in CD86 expression (FIG.
3A) and had no effect on MHC-II or CD40 expression (FIGS. 3B and
3C) in BM-DCs. In contrast, the Hp-16 peptide induced a strong
upregulation of CD86, MHC-II, and CD40 to levels that were
comparable to, or higher than, those generated by LPS stimulation.
Although Hp-106 induced high levels of cytokine secretion in
BM-DCs, this peptide did not significantly enhance the surface
expression of maturation markers. No altered expression in MHC-II,
CD86, or CD40 was detected after exposing BM-DCs to the control
peptide Hp-121 (FIG. 3A-3C).
Example 3
HMGB1-Bx and Select HMGB1 Peptides Induce Functional Maturation of
BM-DCs
Materials and Methods
[0120] Immature BM-DCs that were generated from C57/BL6 mice (FIG.
4A) or Balb/c mice (FIG. 4B) were incubated for 48 h with either
HMGB1-Bx (50 .mu.g/ml), a particular HMGB1 peptide (200 .mu.g/ml),
LPS (100 ng/ml) or were left untreated (medium). T cells were
isolated by negative selection using the mouse SpinSep antibody
cocktail from StemCell Technologies (Vancouver, Calif.), according
to the manufacturer's instructions. The cell purity of the isolated
T cells was routinely .about.99% pure. In order to assess levels of
T cell activation and proliferation, cells were plated at 10.sup.5
cells per well in a round-bottomed 96-well tray at a DC:T cell
ratio of 1:120 for 5 days in the medium described above. The
microcultures were pulsed with (.sup.3H)-thymidine (1 mCi/well) for
the final 8 h of culture. Cell cultures were harvested onto glass
fiber filters with an automated multiple sample harvester and the
amount of isotope incorporation was determined by liquid
scintillation .beta.-emission. Responses are reported as mean cpm
of thymidine incorporation by triplicate cultures (.+-.SEM).
Results
[0121] Mature, cytokine-producing DCs induce T cell activation and
proliferation, leading to the development of adaptive immunity
(Banchereau, J., and R. M. Steinman, Nature 392:245-252 (1998);
Rescigno, M., et al., J. Leukoc. Biol. 61:415-421 (1997)). BM-DCs
that were exposed to HMGB1-Bx, Hp-16 or Hp-106 activated
proliferation of resting allogeneic T cells in a mixed lymphocyte
reaction (FIG. 4A), whereas DCs exposed to Hp-46 or Hp-121 did not
show enhanced T cell stimulatory activity. In order to investigate
whether the functional maturation of DCs caused by exposure to
HMGB1-Bx was strain specific, BM-DCs were generated from Balb/c
mice. As observed with BM-DCs generated from C57/BL6 mice, HMGB1-Bx
treated BM-DCs showed a strong capacity to induce T cell
proliferation (FIG. 4B).
[0122] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0123] While this 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.
Sequence CWU 1
1
191215PRTHomo sapiens 1Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly
Lys Met Ser Ser Tyr1 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 Pro65 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 Ala145 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 277PRTHomo sapiens 2Pro Arg Gly Lys
Met Ser Ser Tyr Ala Phe Phe Val Gln Thr Cys Arg1 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 Thr65
70 75 374PRTHomo sapiens 3Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro
Pro Ser Ala Phe Phe Leu1 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 Tyr65 70 418PRTHomo sapiens 4Met Gly
Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr1 5 10 15
Ala Phe518PRTHomo sapiens 5Tyr Ala Phe Phe Val Gln Thr Cys Arg Glu
Glu His Lys Lys Lys His1 5 10 15 Pro Asp618PRTHomo sapiens 6His Pro
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser1 5 10 15
Glu Arg718PRTHomo sapiens 7Ser Glu Arg Trp Lys Thr Met Ser Ala Lys
Glu Lys Gly Lys Phe Glu1 5 10 15 Asp Met818PRTHomo sapiens 8Glu Asp
Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys1 5 10 15
Thr Tyr918PRTHomo sapiens 9Lys Thr Tyr Ile Pro Pro Lys Gly Glu Thr
Lys Lys Lys Phe Lys Asp1 5 10 15 Pro Asn1018PRTHomo sapiens 10Asp
Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys1 5 10
15 Ser Glu1118PRTHomo sapiens 11Cys Ser Glu Tyr Arg Pro Lys Ile Lys
Gly Glu His Pro Gly Leu Ser1 5 10 15 Ile Gly1218PRTHomo sapiens
12Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys1
5 10 15 Leu Gly1318PRTHomo sapiens 13Ser Ile Gly Asp Val Ala Lys
Lys Leu Gly Glu Met Trp Asn Asn Thr1 5 10 15 Ala Ala1418PRTHomo
sapiens 14Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys
Lys Ala1 5 10 15 Ala Lys1518PRTHomo sapiens 15Thr Ala Ala Asp Asp
Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu1 5 10 15 Lys
Glu1618PRTHomo sapiens 16Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
Ala Tyr Arg Ala Lys Gly1 5 10 15 Lys Pro1718PRTHomo sapiens 17Gly
Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser1 5 10
15 Lys Lys1818PRTHomo sapiens 18Ser Lys Lys Lys Lys Glu Glu Glu Glu
Asp Glu Glu Asp Glu Glu Asp1 5 10 15 Glu Glu1920PRTHomo sapiens
19Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu Glu Glu Asp1
5 10 15 Asp Asp Asp Glu 20
* * * * *