U.S. patent application number 15/191000 was filed with the patent office on 2016-10-13 for hmgn polypeptides as immune enhancers and hmgn antagonists as immune suppressants.
The applicant listed for this patent is The USA, as represented by the Secretary, Dept. of Health and Human Services, The USA, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Michael Bustin, Joost J. Oppenheim, De Yang.
Application Number | 20160296620 15/191000 |
Document ID | / |
Family ID | 41568847 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160296620 |
Kind Code |
A1 |
Yang; De ; et al. |
October 13, 2016 |
HMGN POLYPEPTIDES AS IMMUNE ENHANCERS AND HMGN ANTAGONISTS AS
IMMUNE SUPPRESSANTS
Abstract
A method of enhancing an antigen-specific immune response in a
host comprising administering to the host an HMGN polypeptide
comprising at least one of HMGN1, HMGN3a, HMGN3b, HMGN4, Nsbp1, or
a functional fragment thereof, in an amount effective to enhance an
antigen-specific immune response; as well as a pharmaceutical
composition comprising an HMGN polypeptide comprising at least one
of HMGN1, HMGN3a, HMGN3b, HMGN4, Nsbp1, or a functional fragment
thereof, and an antigen, or nucleic acids encoding such molecules;
and related methods and compositions.
Inventors: |
Yang; De; (Frederick,
MD) ; Oppenheim; Joost J.; (Bethesda, MD) ;
Bustin; Michael; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The USA, as represented by the Secretary, Dept. of Health and Human
Services |
Bethesda |
MD |
US |
|
|
Family ID: |
41568847 |
Appl. No.: |
15/191000 |
Filed: |
June 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13533492 |
Jun 26, 2012 |
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15191000 |
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12509088 |
Jul 24, 2009 |
8227417 |
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13533492 |
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61083781 |
Jul 25, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/489 20180101;
A61K 38/1709 20130101; C12N 5/0639 20130101; A61K 2039/57 20130101;
Y02A 50/30 20180101; A61P 37/06 20180101; Y02A 50/41 20180101; A61K
2039/55516 20130101; C12N 2501/05 20130101; C12N 15/85 20130101;
A61K 39/07 20130101; A61P 37/04 20180101; C12N 2501/23 20130101;
A61K 45/06 20130101; A61K 39/00119 20180801; Y02A 50/491 20180101;
A61K 39/0011 20130101; A61K 39/39 20130101; A61K 2039/53
20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 45/06 20060101 A61K045/06; A61K 39/00 20060101
A61K039/00; C12N 15/85 20060101 C12N015/85; C12N 5/0784 20060101
C12N005/0784 |
Claims
1. A pharmaceutical composition comprising: an HMGN polypeptide
comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ
ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or a
functional fragment thereof, and an antigen.
2. The pharmaceutical composition of claim 1, wherein the HMGN
polypeptide and the antigen are provided as a fusion protein or
conjugate.
3. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition comprises two or more different HMGN
polypeptides, selected from HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID
NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID
NO: 5), or functional fragments thereof.
4. The pharmaceutical composition of claim 1, wherein the HMGN
polypeptide comprises HMGN1 (SEQ ID NO: 1).
5. The pharmaceutical composition of claim 1, wherein the antigen
comprises a tumor antigen.
6. The pharmaceutical composition of claim 1, wherein the antigen
comprises two or more different antigens.
7. The pharmaceutical composition of claim 1, wherein the antigen
is selected from Ig-idiotype of B cell lymphoma; mutant
cyclin-dependent kinase 4 of melanoma; Pmel-17 (gp100) of melanoma;
MART-1 (Melan-A) of melanoma; p15 polypeptide of melanoma;
tyrosinase of melanoma; MAGE 1, 2 and 3 of melanoma, thyroid
medullary, small cell lung cancer, colon and/or bronchial squamous
cell cancer; BAGE of bladder, melanoma, breast, and squamous-cell
carcinoma; gp75 of melanoma; oncofetal antigen of melanoma; muci
mucin of breast, pancreas, and ovarian cancer; GM2 and GD2
gangliosides of melanoma; mutant p53 of carcinoma; mutant ras of
colon cancer; HER21neu proto-onco-gene of breast carcinoma; human
papilloma virus polypeptides of squamous cell cancers of cervix and
esophagus; and combinations thereof.
8. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically active agent.
9. The pharmaceutical composition of claim 8, wherein the
pharmaceutically active agent is a chemotherapeutic agent selected
from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,
doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,
paclitaxel, rituximab, vinblastine, vincristine, bacillus
calmette-guerin (BCG), IL-2, an interferon, GM-CSF, and
trastuzumab.
10. A composition comprising: a nucleic acid encoding an HMGN
polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2),
HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5),
or a functional fragment thereof, and a nucleic acid encoding an
antigen.
11. The composition of claim 10, wherein the nucleic acid encoding
the HMGN polypeptide and the nucleic acid encoding the antigen
comprise a single nucleic acid encoding a fusion protein of the
HMGN polypeptide and the antigen.
12. The composition of claim 10, wherein the nucleic acid encoding
the HMGN polypeptide is at least one sequence selected from any one
of SEQ ID NOs: 6-10, and a degenerate nucleic acid sequence
encoding the same amino acid sequence.
13. A recombinant expression vector comprising a nucleic acid
encoding an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1),
HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4),
Nsbp1 (SEQ ID NO: 5), or a functional fragment thereof, and a
nucleic acid encoding an antigen, which expression vector permits
expression of an HMGN mRNA or polypeptide by a host cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 13/533,492, filed Jun. 26, 2012, which is a
continuation of U.S. patent application Ser. No. 12/509,088, filed
Jul. 24, 2009, issued as U.S. Pat. No. 8,227,417 on Jul. 24, 2012,
which claims the benefit of U.S. Provisional Patent Application No.
61/083,781, filed Jul. 25, 2008, each of which is incorporated by
reference in its entirety herein.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 10,598 Byte
ASCII (Text) file named "710652ST25.TXT," dated Jun. 22, 2016.
BACKGROUND OF THE INVENTION
[0003] HMGN polypeptides belong to the high mobility group (HMG)
family of chromosomal binding peptides. HMGN polypeptides typically
function inside the cell nucleus to bind to DNA and nucleosomes and
regulate the transcription of various genes. HMGN polypeptides also
can be released by peripheral blood mononuclear cells.
[0004] A patient's immune response often plays an important role in
the progression of disease and the effectiveness of medical
treatments for disease. Two types of immune responses can occur in
a patient: a Th-1 pro-inflammatory type and a Th-2
anti-inflammatory type. The Th-1 (cell-mediated) type of immune
response activates T-cells and macrophages, while the Th-2
(antibody-mediated) type of immune response activates B-cells.
[0005] Often, due to the effects of a disease or even the
treatments for a disease, a patient's immune response is diminished
or the immune response is disadvantageously shifted away from a
Th-1 pro-inflammatory type response and towards a Th-2
anti-inflammatory type response. This diminished or Th-2 polarized
immune response is thought to be responsible, at least in part, for
the more rapid progression of disease and reduction in the
effectiveness of some treatments. This is thought to be true, for
example, in cancer patients. In the context of other diseases, a
heightened or Th-1 polarized immune response can be disadvantageous
and at least partly responsible for the progression of the disease
or reduction in the effectiveness of treatment. Thus, there is a
need in the art for methods of modulating an immune response.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] The invention provides a method of enhancing an
antigen-specific immune response in a host comprising administering
to the host an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1),
HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4),
Nsbp1 (SEQ ID NO: 5), or a functional fragment thereof, in an
amount effective to enhance an antigen-specific immune
response.
[0007] In another aspect, the invention provides a method of
enhancing the activation or recruitment of dendritic cells in a
host comprising administering to the host an HMGN polypeptide
comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ
ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or a
functional fragment thereof, in an amount effective to enhance the
activation or recruitment of dendritic cells in a host.
[0008] The invention also provides a method of shifting the
Th-1/Th-2 balance of an immune response of a host towards a Th-1
type immune response comprising administering to the host an HMGN
polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2),
HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5),
or a functional fragment thereof, in an amount effective to shift
the Th-1/Th-2 balance of an immune response of a host towards a
Th-1 type immune response.
[0009] The invention further provides a pharmaceutical composition.
According to one aspect of the invention, the pharmaceutical
composition comprises (a) an HMGN polypeptide comprising HMGN1 (SEQ
ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ
ID NO: 4), Nsbp1 (SEQ ID NO: 5), or a functional fragment thereof,
and (b) an antigen, such as a tumor antigen. According to another
aspect of the invention, the pharmaceutical composition comprises
(a) a nucleic acid that encodes an HMGN polypeptide comprising
HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3),
HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or a functional
fragment thereof, and (b) a nucleic acid that encodes a tumor
antigen.
[0010] In another aspect, the invention provides a method of
suppressing an immune response in a host comprising administering
to the host an HMGN polypeptide antagonist, wherein the HMGN
polypeptide comprises HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2),
HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), or Nsbp1 (SEQ ID NO:
5), in an amount effective to suppress the immune response.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1A illustrates by way of several graphs the
concentration of various cytokines (IL-6, IL-8, IL-12p70, and
TNF.alpha.) in dendritic cell cultures treated with different
concentrations of HMGN1 (SEQ ID NO: 1) or HMGN2. FIG. 1B
illustrates by way of several graphs the concentration of various
cytokines in control dendritic cell cultures (no treatment) and
dendritic cell cultures treated with 1 .mu.g/ml of HMGN1 (SEQ ID
NO: 1) or HMGN2 as a function of time. In FIGS. 1A and 1B, data
from control cultures is referenced by -.largecircle.-, cultures
treated with HMGN1 by - -, and cultures treated with HMGN2 by
-.tangle-solidup.-.
[0012] FIG. 2 presents flow cytometry histograms showing the
proportion of cells (by number) expressing surface costimulatory
molecules (CD80, CD83, and CD 86) and surface major
histocompatibility complex (MHC) molecules (HLA-ABC and HLA-DR) in
control dendritic cell cultures (no treatment or liposaccharide
(LPS)-treated) and dendritic cell cultures treated with HMGN1 (SEQ
ID NO: 1) or HMGN2. Surface expression of CD11c was measured as an
additional control. Surface molecule expression is illustrated as a
function of fluorescence intensity. The open-area curves represent
staining with isotype-matched control antibody, and the shaded area
curves represent staining with antibodies to the various surface
molecules.
[0013] FIG. 3 is a graph of T-cell proliferation (as represented by
tritiated thymidine incorporation) plotted against the ratio of
dendritic cells to T-cells (DC:T) in dendritic cell/mixed
lymphocyte reaction cultures. Treated cultures contained dendritic
cells (DCs) exposed to 1 .mu.g/ml HMGN1 (SEQ ID NO: 1)
(-.tangle-solidup.-), HMGN2 (--), or LPS (-.diamond-solid.-).
Untreated culture (contained sham-treated DCs (- -)) and mixed
lymphocyte reaction culture without dendritic cells
(-.quadrature.-) served as controls.
[0014] FIG. 4 is a graph of dendritic cell migration (number of
cells per high power field (No./HPF)) towards various cytokines
(CCL5, CCL21, and CXCL12) in cultures treated with 1 .mu.g/ml HMGN1
(SEQ ID NO: 1) (diagonal striped bars) or LPS (black bars), and in
untreated culture (contained sham-treated DCs (white bars)). Cell
migration without any cytokine was measured as an additional
control.
[0015] FIG. 5 is a graph of the migration index of subpopulations
of DC cells (CD11c+; CD11c+/CD11b+; CD11c+/B220+; and
CD11c+/CD11b+/B220) in the peritoneal cavity of mice treated with
HMGN1 (SEQ ID NO: 1) (black bars) and control mice treated only
with phosphate buffer (PBS) (white bars).
[0016] FIG. 6A is a graph of cell proliferation (as represented by
tritiated thymidine incorporation) plotted against ovalbumin (OVA)
concentration in cultures of OVA-specific splenocytes harvested
from mice treated with OVA alone (-.largecircle.-) or OVA mixed
with alum (-.diamond-solid.-) or HMGN1 (SEQ ID NO: 1)
(-.tangle-solidup.-). FIG. 6B presents graphs of the concentration
of various cytokines in the same cultures.
[0017] FIG. 7A presents graphs of the concentration of various
cytokines produced in response to in vitro stimulation with OVA by
splenocytes from HMGN1 KO (HMGN1-/-) or wild-type (HMGN1+/+) mice
immunized with OVA in the presence of alum. FIG. 7B presents graphs
of the concentration of various cytokines produced in response to
in vitro stimulation with OVA by splenocytes from HMGN1 KO
(HMGN1-/-) or wild-type (HMGN1+/+) mice immunized with OVA in the
presence of LPS.
[0018] FIG. 8A is a graph of the number of primary anti-anthrax
vaccine adsorbed (AVA) antibodies produced in mice immunized with
AVA alone or in the presence of 1 .mu.g or 5 .mu.g of HMGN1 (SEQ ID
NO: 1). FIG. 8B is a graph of the number of secondary anti-AVA
antibodies produced in mice immunized with AVA alone or in the
presence of 1 .mu.g or 5 .mu.g of HMGN1 (SEQ ID NO: 1).
[0019] FIGS. 9A and 9B are graphs of the amounts of various
cytokines (pg/ml) produced by dendritic cells treated with 0, 0.1,
or 1 .mu.g/ml HMGN1 (SEQ ID NO: 1).
[0020] FIG. 10A is a graph of the concentration of various
cytokines produced by HMGN1 KO (HMGN1-/-) (white bars) or wild-type
(HMGN1+/+) (black bars) mice 24 hours after injection with OVA
alone or OVA in the presence of alum or LPS. FIG. 10B is a graph of
the concentration of various cytokines produced by HMGN1 KO
(HMGN1-/-) (white bars) or wild-type (HMGN1+/+) (black bars) mice
96 hours after injection with OVA alone or OVA in the presence of
alum or LPS.
[0021] FIG. 11 presents a Western blot of untreated or HMGN1 (SEQ
ID NO: 1)-treated (20 or 60 minutes) DC lysates probed with
anti-I-.kappa.B.alpha., anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), anti-phosphorylated p44/42 mitogen-activated
protein kinases (MAPKs), anti-p44/42 MAPKs, anti-phosphorylated p38
MAPK, anti-p38 MAPK, anti-phosphorylated c-Jun N-terminal kinase
(JNK)MAPK, or anti-JNK MAPK antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0022] HMGN polypeptides are members of the high mobility group
(HMG) family of chromosomal binding polypeptides. The HMG family is
subdivided into three subfamilies, each of which has a
characteristic functional sequence motif: HMGB (HMG-box motif),
HMGN (nucleosomal binding domain), and HMGA (AT-hook motif). HMGN
polypeptides include HMGN1 (formerly known as HMG14) (SEQ ID NO:
1), HMGN2, HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ
ID NO: 4), and Nsbp1 (NBD-45) (SEQ ID NO: 5).
[0023] It has been discovered that certain HMGN polypeptides can
enhance an antigen-specific immune response. Thus, the invention
provides methods of using HMGN polypeptides and functional
fragments thereof to enhance an immune response in a host.
[0024] In one embodiment, the invention provides a method of
enhancing an antigen-specific immune response in a host comprising
administering to the host an HMGN polypeptide comprising HMGN1 (SEQ
ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ
ID NO: 4), Nsbp1 (NBD-45) (SEQ ID NO: 5), or a functional fragment
thereof (herein collectively referred to as "an HMGN polypeptide"),
in an amount effective to enhance an antigen-specific immune
response.
[0025] An antigen specific-immune response can be characterized by
the production of lymphocytes that are capable of recognizing and
differentiating the antigen from other antigens and mediating the
destruction of the antigen. An antigen-specific immune response
also can be characterized by the production, maturation,
activation, or recruitment of antigen presenting cells.
[0026] An antigen-specific immune response is enhanced in
accordance with the invention if the immune response to a given
antigen is greater, quantitatively or qualitatively, after
administration of an HMGN polypeptide as compared to the immune
response in the absence of the administration of an HMGN
polypeptide. A quantitative increase in an immune response
encompasses an increase in the magnitude or degree of the response.
The magnitude or degree of an immune response can be measured on
the basis of any number of known parameters, such as an increase in
the level of antigen-specific cytokine production (cytokine
concentration), an increase in the number of lymphocytes activated
(e.g., proliferation of antigen-specific lymphocytes) or recruited,
and/or an increase in the production of antigen-specific antibodies
(antibody concentration), etc. A qualitative increase in an immune
response encompasses any change in the nature of the immune
response that renders it more effective at combating a given
antigen or disease. By way of illustration, an antigen-specific
immune response typically includes two types of immune responses
that occur simultaneously and exist in a relative balance: a Th-1
type response and a Th-2 type response. For the purposes of this
invention, the quality of an immune response is considered enhanced
in quality if the relative balance of the immune response is
shifted towards the Th-1 type immune response and away from the
Th-2 type immune response. Methods of distinguishing and measuring
the relative balance of an immune response are known in the art.
For example, measuring the types and levels of cytokines produced
can distinguish and measure the relative balance of an immune
response. A shift towards the Th-1 type response (e.g., after
administration of an HMGN polypeptide) may be characterized by an
increase in IFN.gamma. and no increase or a reduced increase in
IL-4, IL-5, and/or IL-13 (e.g., as compared to the levels of these
cytokines before administration of an HMGN polypeptide). In other
words, a shift towards a Th-1 type response can be characterized by
an increase in the proportion of IFN.gamma. relative to IL-4, IL-5,
and/or IL-13. Conversely, a shift towards the Th-2 type response
may be characterized by an increase in IL-4, IL-5, and/or IL-13 and
no increase or a reduced increase in IFN.gamma. (e.g., an increase
in the proportion of IL-4, IL-5, and/or IL-13 relative to
IFN.gamma.). Another exemplary method may include measuring the
subtypes of antigen-specific IgG antibodies produced during an
immune response. A higher level (concentration) of IgG2a antibodies
versus IgG1 antibodies suggests a Th1-type immune response.
Conversely, a higher level (concentration) of IgG1 antibodies
versus IgG2a antibodies suggests a Th2-type immune response.
Qualitative and quantitative enhancements in an immune response can
occur simultaneously, and are not mutually exclusive.
[0027] Preferably, the antigen-specific immune response is enhanced
by shifting the Th-1/Th-2 balance of an immune response towards a
Th-1 type response and away from a Th-2 type response, i.e., by
enhancing or increasing the Th-1 type response or by decreasing or
diminishing the Th-2 type response. Enhancing or increasing a
Th1-type immune response may include increasing the production of
cytokines such as IFN.gamma. and/or TNF.alpha. and/or stimulating a
cell-mediated immune response, such as the proliferation and
activation of T-cells and/or macrophages specific for the antigen.
Decreasing the Th2 immune response may include reducing the
antibody-mediated, humoral immune responses and/or the production
of interleukins 4, 5, and 13. In this respect, the invention also
provides a method of shifting the Th-1/Th-2 balance of an immune
response of a host towards a Th-1 type immune response, which
method comprises administering to the host at least one HMGN
polypeptide in an amount effective to shift the Th-1/Th-2 balance
of an immune response of a host towards a Th-1 type immune
response.
[0028] The antigen-specific immune response also can be enhanced by
increasing or enhancing the activation or recruitment of dendritic
cells. Thus, the invention provides, as a related aspect, a method
of enhancing the activation or recruitment of dendritic cells in a
host comprising administering to the host at least one HMGN
polypeptide in an amount effective to enhance the activation or
recruitment of dendritic cells in the host.
[0029] Activation of dendritic cells includes stimulating the
maturation and/or the migration of dendritic cells to a specific
locale (e.g., the site of an antigen or the site of chemotactic
cytokine production, such as CCL2, CCL5, CCL19, CCL20, CCL21, etc.)
and/or stimulating the maturation of dendritic cells. The
activation of dendritic cells can be detected or measured by the
production of cytokines associated with the activation of dendritic
cells. In particular, the HMGN polypeptide (or a functional
fragment thereof) may stimulate the dendritic cells to produce
cytokines such as, for example, any or all of interleukin (IL)-6,
IL-2, IL-8, IL-12, (e.g., IL-12p70), IL-1 (e.g., IL-1.beta.),
IL-10, IL-18, IL-23, tumor necrosis factors (TNF) (e.g.,
TNF.alpha.), and/or chemokines (e.g., keratinocyte chemoattractant
(KC), CXCL8, CCL1, CCL2, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18,
CCL20, and/or CCL22). Activation of dendritic cells can also be
detected or measured by the phosphorylation of mitogen-activated
protein kinases (MAPKs) associated with the activation of dendritic
cells. In particular, the HMGN polypeptide (or a functional
fragment thereof) may stimulate the phosphorylation of MAPKs such
as, for example, any or all of p44/42 MAPKs, p38 MAPKs, and/or
c-Jun N-terminal kinase (JNK) MAPKs. In addition, the activation of
dendritic cells can be detected or measured by the activation of
nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-.kappa.B). In particular, the HMGN polypeptide (or a functional
fragment thereof) may stimulate the activation of NF-.kappa.B
and/or a decrease of I-.kappa.B.alpha.. Maturation of dendritic
cells can be detected or measured on the basis of the expression of
surface molecules that appear on mature dendritic cells. For
example, mature dendritic cells typically express receptors that
enable them to respond to chemokines produced by the lymph node
(e.g., CCR7) and costimulatory (e.g., CD80, CD83, and CD86) and MHC
(e.g., HLA-ABC and HLA-DR) molecules that assist in activating
T-cells. Maturation of dendritic cells also can be detected by a
cell shape having veils and elongated dendrites, increased motility
toward chemokines (e.g., CCL19 and CCL21), or reduced capacity for
endocytosis. Maturation of dendritic cells also can be detected
indirectly by measuring the capacity of dendritic cells to
stimulate the proliferation or differentiation of naive T-cells.
Recruitment of dendritic cells can be measured or detected by the
movement of dendritic cells to a given locale. Assays for measuring
or detecting an increase in the activation and/or recruitment of
dendritic cells are known in the art and described herein.
[0030] The HMGN polypeptides (including functional fragments
thereof) can be obtained by methods known in the art. Suitable
methods of de novo synthesizing polypeptides are described in
references, such as Chan et al., Fmoc Solid Phase Peptide
Synthesis, Oxford University Press, Oxford, United Kingdom, 2005;
Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,
Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University
Press, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752.
Also, HMGN polypeptides (including functional fragments) and
antigens can be recombinantly produced using the nucleic acids
described herein and standard recombinant methods. See, for
instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,
3.sup.rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
2001; and Ausubel et al., Current Protocols in Molecular Biology,
Greene Publishing Associates and John Wiley & Sons, N Y, 1994.
Further, the HMGN polypeptides (including functional fragments
thereof) can be isolated and/or purified from a natural source,
e.g., a human. Methods of isolation and purification are well-known
in the art. In this respect, the HMGN polypeptides are exogenous
and can be synthetic, recombinant, or of natural origin.
[0031] The functional fragment of the HMGN polypeptide can comprise
any contiguous part of the HMGN polypeptide that retains a relevant
biological activity of the HMGN polypeptide, e.g., enhances an
antigen-specific immune response. Any given fragment of an HMGN
polypeptide can be tested for such biological activity using
methods described herein or otherwise known in the art. For
example, the functional fragment can comprise, consist essentially
of, or consist of the N-terminal nucleosomal binding domain (NBD)
of the HMGN polypeptide (e.g., the sequence from .sup.14Lys to
.sup.49Lys of HMGN1 (SEQ ID NO: 11), the sequence from .sup.20Lys
to .sup.49Lys of HMGN3a/3b (SEQ ID NO: 12), the sequence from
.sup.18Lys to .sup.47Lys of HMGN4 (SEQ ID NO: 13), and/or the
sequence from .sup.17Lys to .sup.46Lys of Nsbp1 (SEQ ID NO: 14)).
In reference to the parent HMGN polypeptide, the functional
fragment preferably comprises, for instance, about 10% or more, 25%
or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or
more, or even 95% or more of the parent HMGN polypeptide.
[0032] The HMGN polypeptides (including functional fragments) can
be glycosylated, amidated, carboxylated, phosphorylated,
esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or
converted into an acid addition salt and/or optionally dimerized or
polymerized, or conjugated. Suitable pharmaceutically acceptable
acid addition salts include those derived from mineral acids, such
as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,
and sulphuric acids, and organic acids, such as tartaric, acetic,
citric, malic, lactic, fumaric, benzoic, glycolic, gluconic,
succinic, and arylsulphonic acids, for example, p-toluenesulphonic
acid.
[0033] Of course, the method of the invention can comprise
administering two or more HMGN polypeptides or functional fragment
thereof, any of which may be the same or different from one
another. Furthermore, the HMGN polypeptide or functional fragment
thereof can be provided as part of a larger polypeptide construct.
For instance, the HMGN polypeptide or functional fragment thereof
can be provided as a fusion protein comprising an HMGN polypeptide
or functional fragment along with other amino acid sequences or a
nucleic acid encoding same. By way of further illustration, the
HMGN polypeptide or functional fragment can be provided by two or
more fragments of an HMGN polypeptide (e.g., two or more NBD
domains, or at least one of each of the NBD domains), with or
without a linking amino acid sequence and/or flanking sequences.
The HMGN polypeptide or fragment thereof also can be provided as
part of a conjugate or nucleic acid encoding same. Conjugates, as
well as methods of synthesizing conjugates in general, are known in
the art (See, for instance, Hudecz, F., Methods Mol. Biol. 298:
209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415
(2005)).
[0034] The antigen can be any antigen against which an
antigen-specific immune response is desired. In some embodiments,
the antigen is a microbial antigen. The microbial antigen can be a
bacterial (e.g., anthrax, tuberculosis, etc.) antigen or a viral
(e.g., influenza, human immunodeficiency virus (HIV), etc.)
antigen. Microbial antigens are molecules (e.g., polypeptide,
lipid, carbohydrate, etc.) that are uniquely expressed by microbes,
or greatly over-expressed by microbes as compared to non-microbes,
such that an immune response to the antigen results in the more
rapid destruction of the microbe as compared to non-microbes.
[0035] Preferably, the antigen-specific immune response is an
immune response to a tumor antigen. Tumor antigens are molecules
(e.g., polypeptide, lipid, carbohydrate, etc.) that are uniquely
expressed by tumor cells, or greatly over-expressed by tumor cells
as compared to non-tumor cells, such that an immune response to the
antigen results in the more rapid destruction of tumor cells as
compared to normal (non-cancerous) cells.
[0036] The tumor antigen can be an antigen expressed by any cell of
any cancer or tumor. For example, the tumor antigen can be an
antigen expressed by any cell of acute lymphocytic cancer, acute
myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain
cancer, breast cancer, cancer of the anus, anal canal, or
anorectum, cancer of the eye, cancer of the intrahepatic bile duct,
cancer of the joints, cancer of the neck, gallbladder, or pleura,
cancer of the nose, nasal cavity, or middle ear, cancer of the oral
cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic
myeloid cancer, colon cancer, uterine cancer, esophageal cancer,
cervical cancer, gastrointestinal carcinoid tumor, lymphoid and
other hematopoietic tumors, Hodgkin lymphoma, B cell lymphoma,
bronchial squamous cell cancer, hypopharynx cancer, kidney cancer,
larynx cancer, liver cancer, pancreatic cancer, carcinoma, lung
cancer, malignant mesothelioma, melanoma, multiple myeloma,
nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic cancer, peritoneum, omentum, and mesentery cancer,
pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g.,
renal cell carcinoma (RCC)), small intestine cancer, soft tissue
cancer, stomach cancer, testicular cancer, thyroid cancer, ureter
cancer, and urinary bladder cancer.
[0037] More specific examples of tumor antigens include
polypeptides such as Ig-idiotype of B cell lymphoma, mutant
cyclin-dependent kinase 4 of melanoma, Pmel-17 (gp100) of melanoma,
MART-1 (Melan-A) of melanoma, p15 polypeptide of melanoma,
tyrosinase of melanoma, MAGE 1, 2 and 3 of melanoma, thyroid
medullary, small cell lung cancer, colon and/or bronchial squamous
cell cancer, BAGE of bladder, melanoma, breast, and squamous-cell
carcinoma, gp75 of melanoma, oncofetal antigen of melanoma;
carbohydrate/lipids such as muci mucin of breast, pancreas, and
ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes
such as mutant p53 of carcinoma, mutant ras of colon cancer and
HER21neu proto-onco-gene of breast carcinoma; viral products such
as human papilloma virus polypeptides of squamous cell cancers of
cervix and esophagus.
[0038] A method of the invention may further comprise administering
an antigen to the host, especially a tumor antigen. The antigen can
be any of those discussed above with respect to the
antigen-specific immune response. Of course, two or more different
antigens can be administered to the host.
[0039] When an antigen is administered in connection with a method
of the invention, the antigen and HMGN polypeptide (or functional
fragment thereof) can be administered simultaneously (as a single
composition or in different compositions) or sequentially in any
order. The methods may include administering the antigen first,
followed by the HMGN polypeptide (or functional fragment thereof),
or the method may include administering the HMGN polypeptide (or
functional fragment thereof) first, followed by the antigen.
Regardless of the order of the administration of the HMGN
polypeptide (or functional fragment thereof) and the antigen, the
HMGN polypeptide (or functional fragment thereof) and the antigen
are preferably administered in close enough succession to enhance
an immune response against the antigen.
[0040] The HMGN polypeptide (or functional fragment thereof) and
the antigen also can be part of a fusion protein. The fusion
protein can comprise one or more HMGN polypeptides (or functional
fragments thereof) and/or one or more antigens. Suitable methods of
making fusion proteins are known in the art, and include, for
example, recombinant methods. See, for instance, Choi et al., Mol.
Biotechnol. 31: 193-202 (2005). In other embodiments, the HMGN
polypeptide, including any of the functional fragments thereof, may
be provided as a conjugate with the antigen. Conjugates, as well as
methods of synthesizing conjugates in general, are known in the art
(See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223
(2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).
[0041] The HMGN polypeptide or fragment thereof and the antigen,
when used, can be administered to the host by administering a
nucleic acid encoding such molecules to the host. "Nucleic acid" as
used herein includes "polynucleotide," "oligonucleotide," and
"nucleic acid molecule," and generally means a polymer of DNA or
RNA, which can be single-stranded or double-stranded, synthesized
or obtained (e.g., isolated and/or purified) from natural sources,
which can contain natural, non-natural or altered nucleotides, and
which can contain a natural, non-natural or altered internucleotide
linkage, such as a phosphoroamidate linkage or a phosphorothioate
linkage, instead of the phosphodiester found between the
nucleotides of an unmodified oligonucleotide.
[0042] Nucleic acids encoding the HMGN polypeptides and antigens
discussed herein are known in the art (e.g., SEQ ID NOs: 6-10 and
degenerate nucleic acid sequences encoding the same amino acid
sequences), and can be constructed based on chemical synthesis
and/or enzymatic ligation reactions using procedures known in the
art. See, for example, Sambrook et al., supra, and Ausubel et al.,
supra. For example, a nucleic acid can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed upon hybridization (e.g., phosphorothioate derivatives and
acridine substituted nucleotides).
[0043] The nucleic acids can be incorporated into a recombinant
expression vector. For purposes herein, the term "recombinant
expression vector" means a genetically-modified oligonucleotide or
polynucleotide construct that permits the expression of an mRNA or
polypeptide by a host cell, when the construct comprises a
nucleotide sequence encoding the mRNA or polypeptide, and the
vector is contacted with the cell under conditions sufficient to
have the mRNA or polypeptide expressed within the cell. The vectors
are not naturally-occurring as a whole. However, parts of the
vectors can be naturally-occurring. The recombinant expression
vectors can comprise any type of nucleotides, including, but not
limited to DNA and RNA, which can be single-stranded or
double-stranded, synthesized or obtained in part from natural
sources, and which can contain natural, non-natural or altered
nucleotides. The recombinant expression vectors can comprise
naturally-occurring or non-naturally-occurring internucleotide
linkages, or both types of linkages. Preferably, the non-naturally
occurring or altered nucleotides or internucleotide linkages does
not hinder the transcription or replication of the vector.
[0044] The recombinant expression vector can be any suitable
recombinant expression vector, and can be used to transform or
transfect any suitable host. Suitable vectors include those
designed for propagation and expansion or for expression or both,
such as plasmids and viruses. The vector can be of the pUC series
(Fermentas Life Sciences), the pBluescript series (Stratagene,
LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX
series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series
(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as
.lamda.GT10, .lamda.GT11, .lamda.ZapII (Stratagene), .lamda.EMBL4,
and .lamda.NM1149, also can be used. Examples of plant expression
vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19
(Clontech). Examples of animal expression vectors include pEUK-Cl,
pMAM and pMAMneo (Clontech). Preferably, the recombinant expression
vector is a viral vector, e.g., a retroviral vector.
[0045] The recombinant expression vectors can be prepared using
standard recombinant DNA techniques described in, for example,
Sambrook et al., supra, and Ausubel et al., supra. Constructs of
expression vectors, which are circular or linear, can be prepared
to contain a replication system functional in a prokaryotic or
eukaryotic host cell. Replication systems can be derived, e.g.,
from ColE1, 2.mu. plasmid, SV40, bovine papilloma virus, and the
like.
[0046] Desirably, the recombinant expression vector comprises
regulatory sequences, such as transcription and translation
initiation and termination codons, which are specific to the type
of host (e.g., bacterium, fungus, plant, or animal) into which the
vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA- or RNA-based.
[0047] The recombinant expression vector can include one or more
marker genes, which allow for selection of transformed or
transfected hosts. Marker genes include biocide resistance, e.g.,
resistance to antibiotics, heavy metals, etc., complementation in
an auxotrophic host to provide prototrophy, and the like. Suitable
marker genes for the inventive expression vectors include, for
instance, neomycin/G418 resistance genes, hygromycin resistance
genes, histidinol resistance genes, tetracycline resistance genes,
and ampicillin resistance genes.
[0048] The recombinant expression vector can comprise a native or
nonnative promoter and/or stop codon operably linked to the
nucleotide sequence encoding the HMGN polypeptide (including
functional fragments thereof), or to the nucleotide sequence which
is complementary to the nucleotide sequence encoding the HMGN
polypeptide or functional fragment thereof. The selection of stop
codons and promoters, e.g., strong, weak, inducible,
tissue-specific and developmental-specific, is within the ordinary
skill of the artisan. Similarly, the combining of a nucleotide
sequence with a stop codon and a promoter is also within the skill
of the artisan. The promoter can be a non-viral promoter or a viral
promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter,
an RSV promoter, and a promoter found in the long-terminal repeat
of the murine stem cell virus.
[0049] The HMGN polypeptides (including functional fragments
thereof) and nucleic acids encoding such peptides can be of
synthetic or natural origin, and can be isolated or purified to any
degree. The terms "isolated" and "purified" as used herein means
having been increased in purity, wherein "purity" is a relative
term, and not to be necessarily construed as absolute purity. For
example, the purity can be at least about 50%, can be greater than
60%, 70% or 80%, or can be 100%.
[0050] The methods described herein may be used for any purpose,
e.g., the treatment or prevention of disease, especially cancer.
Exemplary cancers that may be treated or prevented using the
methods described herein may include any of those discussed above
with respect to the tumor antigens.
[0051] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the inventive methods can provide any amount of
any level of treatment or prevention of cancer in a mammal.
Furthermore, the treatment or prevention provided by the inventive
method can include treatment or prevention of one or more
conditions or symptoms of the disease, e.g., cancer, being treated
or prevented. Also, for purposes herein, "prevention" can encompass
delaying the onset of the disease, or a symptom or condition
thereof. With respect to the inventive methods, the cancer can be
any cancer, including any of the cancers associated with any of the
tumor antigens described herein.
[0052] For purposes of the invention, the amount or dose of the
HMGN material administered should be sufficient to effect the
desired biological response, e.g., a therapeutic or prophylactic
response, in the subject or animal over a reasonable time frame.
The dose will be determined by the efficacy of the particular HMGN
material and the condition of the host (e.g., human), as well as
the body weight of the host (e.g., human) to be treated. The dose
of the HMGN material also will be determined by the existence,
nature and extent of any adverse side effects that might accompany
the administration of a particular HMGN material. Typically, the
attending physician will decide the dosage of the HMGN material
with which to treat each individual patient, taking into
consideration a variety of factors, such as age, body weight,
general health, diet, sex, HMGN material to be administered, route
of administration, and the severity of the condition being
treated.
[0053] The host referred to in the inventive methods can be any
host capable of exhibiting an antigen-specific immune response.
Preferably, the host is a mammal. As used herein, the term "mammal"
refers to any mammal, including, but not limited to, mammals of the
order Rodentia, such as mice and hamsters, and mammals of the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Carnivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simoids (monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human. The host can be
non-diseased, a host afflicted with a disease, such as cancer, or a
host predisposed to a disease, such as cancer.
[0054] The invention also provides a pharmaceutical composition
comprising (a) an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1),
HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4),
Nsbp1 (NBD-45) (SEQ ID NO: 5), or functional fragment thereof, and
(b) an antigen, especially a tumor antigen. Alternatively, the
pharmaceutical composition comprises (a) a nucleic acid encoding an
HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID
NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (NBD-45)
(SEQ ID NO: 5), or functional fragment thereof, and (b) a nucleic
acid encoding an antigen, especially a tumor antigen. The
pharmaceutical composition can, of course, comprise more than one
HMGN polypeptide or fragment thereof (e.g., two or more different
HMGN polypeptides) or one or more nucleic acids encoding more than
one HMGN polypeptide or fragment thereof (e.g., two or more
different HMGN polypeptides). Alternatively or in addition, the
pharmaceutical composition can comprise more than one tumor antigen
(e.g., two or more different antigens) or one or more nucleic acids
encoding more than one tumor antigen (e.g., two or more different
antigens). All other features of the HMGN polypeptides (including
functional fragments thereof), antigens, tumor antigens, and
nucleic acids are as described with respect to the methods of the
invention.
[0055] The pharmaceutical composition can comprise other active
ingredients in addition to the HMGN polypeptide, fragment thereof,
and tumor antigen. For example, the pharmaceutical composition can
comprise other pharmaceutically active agents or drugs, such as a
chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin,
cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,
hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,
vincristine, etc.; biological response modifiers, e.g., bacillus
calmette-guerin (BCG), etc.; cytokines, e.g., IL-2, IFNs, GM-CSF,
etc.; and/or antibodies, e.g., trastuzumab (Herceptin.RTM.,
available from Genentech, South San Francisco, Calif., U.S.A.),
etc.
[0056] The pharmaceutical composition typically will comprise a
carrier. Preferably, the carrier is a pharmaceutically acceptable
carrier. With respect to pharmaceutical compositions, the carrier
can be any of those conventionally used and is limited only by
chemico-physical considerations, such as solubility and lack of
reactivity with the active compound(s), and by the route of
administration. The pharmaceutically acceptable carriers described
herein, for example, vehicles, excipients, and diluents, are
well-known to those skilled in the art and are readily available to
the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the active agent(s) and
one which has no detrimental side effects or toxicity under the
conditions of use. The choice of carrier will be determined in part
by the particular compounds used in the pharmaceutical composition,
as well as by the particular method used to administer the HMGN
material.
[0057] The following formulations for oral, intravenous,
intramuscular, subcutaneous, or intraperitoneal administration are
exemplary and are in no way limiting. More than one route can be
used to administer the HMGN materials and/or tumor antigen, and in
certain instances, a particular route can provide a more immediate
and more effective response than another route.
[0058] Oral formulations may include any suitable carrier. For
example, formulations suitable for oral administration may comprise
suitable carriers, such as lactose, sucrose, starch, talc magnesium
stearate, crystalline cellulose, methyl cellulose, carboxymethyl
cellulose, glycerin, sodium alginate or gum arabic among
others.
[0059] Intravenous, intramuscular, subcutaneous, or intraperitoneal
formulations may include any suitable carrier. For example,
formulations suitable for intravenous, intramuscular, subcutaneous,
or intraperitoneal administration may comprise sterile aqueous
solutions of the HMGN polypeptide (or functional fragment thereof)
and/or the tumor antigen with solutions which are preferably
isotonic with the blood of the recipient. Such formulations may be
prepared by dissolving the HMGN polypeptide (or functional fragment
thereof) and/or the tumor antigen in water containing
physiologically compatible substances such as sodium chloride (e.g.
0.1-2.0M), glycine, and the like, and having a buffered pH
compatible with physiological conditions to produce an aqueous
solution, and rendering said solution sterile.
[0060] For purposes of the invention, the amount or concentration
of the HMGN polypeptide or fragment thereof, tumor antigen, and
other optional active ingredients used in the pharmaceutical
composition should be sufficient to effect a desired biological
response, e.g., a therapeutic or prophylactic response, in the
subject or animal using a reasonable dosage regimen over a
reasonable time frame. For example, the concentration of the HMGN
polypeptide or fragment thereof, tumor antigen, and other optional
active ingredients should be sufficient to enhance an
antigen-specific immune response as defined herein with respect to
the methods of the invention.
[0061] The pharmaceutical compositions of the invention may be used
for any purpose, but are thought to be especially useful in
conjunction with the methods of the invention and for the treatment
or prevention of disease, such as cancer. Exemplary cancers that
may be treated or prevented include acute lymphocytic cancer, acute
myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain
cancer, breast cancer, cancer of the anus, anal canal, or
anorectum, cancer of the eye, cancer of the intrahepatic bile duct,
cancer of the joints, cancer of the neck, gallbladder, or pleura,
cancer of the nose, nasal cavity, or middle ear, cancer of the oral
cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic
myeloid cancer, colon cancer, uterine cancer, esophageal cancer,
cervical cancer, gastrointestinal carcinoid tumor, lymphoid and
other hematopoietic tumors, Hodgkin lymphoma, B cell lymphoma,
bronchial squamous cell cancer, hypopharynx cancer, kidney cancer,
larynx cancer, liver cancer, pancreatic cancer, carcinoma, lung
cancer, malignant mesothelioma, melanoma, multiple myeloma,
nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic cancer, peritoneum, omentum, and mesentery cancer,
pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g.,
renal cell carcinoma (RCC)), small intestine cancer, soft tissue
cancer, stomach cancer, testicular cancer, thyroid cancer, ureter
cancer, and urinary bladder cancer.
[0062] The pharmaceutical compositions of the invention may
advantageously be nontoxic. Without being bound to any particular
theory, it is believed that because the HMGN polypeptides are
endogenous molecules, the administration of an HMGN polypeptide may
not cause toxic effects in mammals, particularly, humans.
[0063] The invention also provides a method of suppressing an
antigen-specific immune response in a host. The method comprises
administering to the host an HMGN polypeptide antagonist, wherein
the HMGN polypeptide comprises HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID
NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), or Nsbp1 (SEQ
ID NO: 5), in an amount effective to suppress the immune
response.
[0064] An immune response is suppressed in accordance with the
invention if the immune response is diminished, quantitatively or
qualitatively, after administration of an HMGN polypeptide
antagonist as compared to the immune response in the absence of the
administration of an HMGN polypeptide antagonist. A quantitative
decrease in an immune response encompasses a decrease in the
magnitude or degree of the response. The magnitude or degree of an
immune response can be measured on the basis of any number of known
parameters, such as a decrease in the level of cytokine (e.g.,
antigen-specific cytokine) production (cytokine concentration), a
decrease in the number of lymphocytes activated (e.g.,
proliferation of lymphocytes (e.g., antigen-specific lymphocytes))
or recruited, and/or a decrease in the production of antibodies
(antigen-specific antibodies) (antibody concentration), etc. A
qualitative decrease in an immune response encompasses any change
in the nature of the immune response that renders it less effective
at mediating the destruction of a given antigen. For the purposes
of this invention, the quality of an immune response is considered
diminished if the relative balance of the immune response is
shifted towards the Th-2 type immune response and away from the
Th-1 type immune response. The relative balance of an immune
response may be distinguished and measured by methods known in the
art and as described herein. For example, a shift toward the Th-2
type response may be characterized by an increase in IL-4, IL-5,
and/or IL-13 and no increase or a reduced increase in IFN.gamma..
Conversely, a shift toward the Th-1 type response may be
characterized by an increase in IFN.gamma. and no increase or a
reduced increase in IL-4, IL-5, and/or IL-13. Another exemplary
method may include measuring the subtypes of antigen-specific IgG
antibodies produced during an immune response. A lower level
(concentration) of IgG2a antibodies versus IgG1 antibodies suggests
a Th2-type immune response. Conversely, a lower level
(concentration) of IgG1 antibodies versus IgG2a antibodies suggests
a Th1-type immune response. Qualitative and quantitative
diminishment of an immune response can occur simultaneously, and
are not mutually exclusive.
[0065] Preferably, the immune response is suppressed by shifting
the Th-1/Th-2 balance of an immune response towards a Th-2 type
response and away from a Th-1 type response, i.e., by suppressing
or decreasing the Th-1 type response or by increasing or enhancing
the Th-2 type response. Suppressing or decreasing a Th1-type immune
response may include decreasing the production of cytokines such as
IFN.gamma. and/or TNF.alpha. and/or reducing a cell-mediated immune
response, such as the proliferation and activation of T-cells
and/or macrophages specific for the antigen. Decreasing the Th1
immune response may include increasing the antibody-mediated,
humoral immune responses and/or the production of interleukins 4,
5, and 13.
[0066] The immune response also can be suppressed by decreasing or
suppressing the activation or recruitment of dendritic cells.
Suppressing the activation of dendritic cells includes reducing the
maturation and/or the migration of dendritic cells, e.g., to a
specific locale (e.g., the site of an antigen or the site of
chemotactic cytokine production, such as CCL2, CCL5, CCL19, CCL20,
CCL21, etc.). Suppressing the activation of dendritic cells can be
measured by the lack of production of cytokines associated with the
activation of dendritic cells. In particular, the HMGN polypeptide
antagonist may suppress the dendritic cell production of cytokines
such as, for example, any or all of interleukin (IL)-6, IL-8,
IL-12, (e.g., IL-12p70), IL-1 (e.g., IL-1.beta.), IL-10, IL-18,
IL-23, tumor necrosis factors (TNF) (e.g., TNF.alpha.), and/or
chemokines (e.g., CXCL8, CCL1, CCL2, CCL5, CCL7, CCL8, CCL13,
CCL17, CCL18, CCL20, and/or CCL22). The lack of mature dendritic
cells, or a reduction in mature dendritic cells, can be detected or
measured on the basis of the lack of expression of surface
molecules that appear on mature dendritic cells. For example,
immature dendritic cells typically do not express receptors that
enable them to respond to chemokines produced by the lymph node
(e.g., CCR7) or costimulatory (e.g., CD80, CD83, and CD86) or MHC
(e.g., HLA-ABC and HLA-DR) molecules that assist in activating
T-cells. Immature dendritic cells also can be detected by a cell
shape lacking veils and elongated dendrites, decreased motility
toward chemokines (e.g., CCL19 and CCL21), or increased capacity
for endocytosis. Immature dendritic cells can also be detected
indirectly by measuring the inability of dendritic cells to
stimulate the proliferation or differentiation of naive T-cells.
The absence of recruitment of dendritic cells can be measured or
detected by a lack of movement of dendritic cells to a given
locale. Assays for measuring or detecting a decrease in the
activation and/or recruitment of dendritic cells are known in the
art and described herein.
[0067] The HMGN antagonist can be any agent that inhibits the
biological activity of an HMGN polypeptide. Inhibition of an HMGN
polypeptide may be characterized by suppression of an immune
response in any of the ways described herein. The HMGN antagonists
include agents that bind to the HMGN polypeptide or functional
fragment thereof (e.g., the NBD of the HMGN polypeptide), thereby
inhibiting its function, as well as agents that compete with the
HMGN polypeptide or functional fragment thereof (e.g., the NBD of
the HMGN polypeptide) for the native HMGN binding site. By way of
illustration, the HMGN antagonist can be an antibody or antibody
fragment, an antisense nucleotide, or a chemical inhibitor (e.g.,
small molecule or peptide inhibitor).
[0068] Anti-HMGN antibodies and antibody fragments can be
monoclonal or polyclonal. Anti-HMGN antibodies and antibody
fragments can be prepared using the HMGN proteins disclosed herein
and routine techniques. Examples of such antibodies or antibody
fragments include those specific to a functional domain of HMGN
(e.g., nucleosomal binding domain).
[0069] Antisense nucleic acid (e.g., RNA or DNA) include, for
example, interfering nucleic acids such as RNAi and siRNA
molecules. Such antisense nucleic acids are commercially available
and can be prepared using the nucleic acid sequences encoding the
HMGN polypeptides disclosed herein and routine techniques.
[0070] Chemical inhibitors of HMGN include small molecules and
peptides that bind the HMGN polypeptide or functional fragment
thereof or compete with the HMGN polypeptide or functional fragment
thereof for its native binding site. Suitable inhibitors can
include, for example, a non-active fragment or mutant of an HMGN
polypeptide. Chemical inhibitors of HMGN can be identified using
routine techniques. For example, chemical inhibitors can be tested
in binding assays to identify molecules and peptides that bind to a
given HMGN polypeptide or functional fragment thereof with
sufficient affinity to inhibit HMGN biological function. Also,
competition assays can be performed to identify small-molecules and
peptides that compete with HMGN or functional fragment thereof for
binding to its native binding site. Such techniques could be used
in conjunction with mutagenesis of the HMGN polypeptide or
functional fragment thereof itself, and/or with high-throughput
screens of known chemical inhibitors.
[0071] The methods of suppressing an immune response described
herein may be used for any purpose, e.g., the treatment or
prevention of a disease associated with a heightened or Th-1
polarized immune response, particularly any disease that may be
effectively treated or prevented by shifting the Th-1/Th-2 balance
of an immune response away from a Th1-type response and toward a
Th2-type response. Exemplary diseases that may be treated or
prevented using the methods of suppressing an immune response
described herein include parasitic infections (e.g., Giardia
intestinalis, Trichomonas vaginalis, Cryptosporidium, Toxoplasma
gondii and Leishmania major) and inflammatory or autoimmune
disorders (e.g., atherosclerosis; asthma; lung fibrosis;
bronchitis; respiratory distress syndrome; obstructive pulmonary
disease; allergies; multiple sclerosis; dermatitis; psoriasis;
gastroenteritis; colitis (e.g., ulcerative colitis); Crohn's
disease; cystic fibrosis; celiac disease; inflammatory bowel
disease; conjunctivitis; uveitis; autoimmune kidney disease;
diabetic nephropathy; cachexia; coronary restenosis; sinusitis,
cystitis; urethritis; serositis; uremic pericarditis; cholecystis;
vaginitis; drug reactions; hepatitis; pelvic inflammatory disease;
multiple myeloma; vitiligo; alopecia; Addison's disease;
Hashimoto's disease; Graves disease; atrophic gastritis/pernicious
anemia; acquired hypogonadism/infertility; hypoparathyroidism;
multiple sclerosis; Myasthenia gravis; Coombs positive hemolytic
anemia; systemic lupus erthymatosis; Siogren's syndrome, rheumatoid
arthritis; endotoxemia; and immune mediated (type-1) diabetes).
[0072] For purposes of the invention, the amount or dose of the
HMGN polypeptide antagonist administered should be sufficient to
effect the desired biological response, e.g., a therapeutic or
prophylactic response, in the subject or animal over a reasonable
time frame. The dose will be determined by the efficacy of the
particular HMGN polypeptide antagonist and the condition of the
host (e.g., human), as well as the body weight of the host (e.g.,
human) to be treated. The dose of the HMGN polypeptide antagonist
also will be determined by the existence, nature and extent of any
adverse side effects that might accompany the administration of a
particular HMGN polypeptide antagonist. Typically, the attending
physician will decide the dosage of the HMGN polypeptide antagonist
with which to treat each individual patient, taking into
consideration a variety of factors, such as age, body weight,
general health, diet, sex, HMGN polypeptide antagonist to be
administered, route of administration, and the severity of the
condition being treated.
[0073] Carriers, formulations, and routes of administration of the
HMGN polypeptide antagonist may be any of those described herein
for the administration of the HMGN polypeptide.
[0074] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0075] This example demonstrates that HMGN1 (SEQ ID NO: 1) induces
dendritic cells to produce cytokines in a dose-dependent
manner.
[0076] Human dendritic cells were cultured for 24 hours in RPMI
1640 medium, which contains 10% fetal bovine serum, 50 ng/mL
recombinant human (rh) GM-CSF, 50 ng/mL rhIL-4. The cultures were
treated with either HMGN1 (SEQ ID NO: 1) or HMGN2 at concentrations
of 0, 0.2, 1, or 5 .mu.g/mL. Untreated culture (sham) served as a
control. The supernatants of the dendritic cell cultures were
assayed for IL-6, IL-8, IL-12p70, and TNF.alpha. concentration by
cytokine array. Experiments were repeated three times and the
average (mean.+-.SD) determined. The results are presented in FIG.
1A.
[0077] As shown in FIG. 1A, HMGN1 (SEQ ID NO: 1) stimulated the
production of IL-6, IL-8, IL-12p70, and TNF.alpha. in dendritic
cells a dose-dependent manner, whereas HMGN2 and untreated cultures
showed no significant production of these cytokines. These results
support the use of HMGN1 (SEQ ID NO: 1) to activate and/or recruit
dendritic cells and enhance an immune response.
Example 2
[0078] This example demonstrates that HMGN1 (SEQ ID NO: 1) induces
dendritic cells to produce cytokines in a time-dependent
manner.
[0079] Human dendritic cells were cultured for 24 hours in RPMI
1640 medium. The cultures were treated with 1 .mu.g/mL of HMGN1
(SEQ ID NO: 1) or HMGN2, and the supernatents were analyzed by
cytokine array for IL-6, IL-8, IL-12p70, and TNF.alpha.
concentration at 6, 24, and 48 hours. Untreated culture (sham)
served as a control. Experiments were repeated three times and the
average (mean.+-.SD) determined. The results are presented in FIG.
1B.
[0080] As shown in FIG. 1B, HMGN1 (SEQ ID NO: 1) stimulates the
production of IL-6, IL-8, IL-12p70, and TNF.alpha. in a
time-dependent manner, whereas HMGN2 and untreated cultures showed
no significant production of these cytokines. These results support
the use of HMGN1 (SEQ ID NO: 1) to activate and/or recruit
dendritic cells and enhance an immune response.
Example 3
[0081] This example demonstrates that HMGN1 (SEQ ID NO: 1)
upregulates dendritic cell expression of surface costimulatory
molecules and surface MHC molecules.
[0082] Human dendritic cells were cultured for 48 hours at
37.degree. C. in a CO.sub.2 incubator in RPMI 1640 medium. Cultures
were treated with 1 .mu.g/mL of HMGN1 (SEQ ID NO: 1) or HMGN2.
Culture treated with 1 .mu.g/mL lipopolysaccharide (LPS) and
untreated culture (sham) served as positive and negative controls,
respectively. The dendritic cells were immunostained and analyzed
for the expression of surface molecules by flow cytometry. The
results are presented in FIG. 2, wherein open-area curves represent
staining with isotype-matched control antibody, and shaded-area
curves represent staining with antibodies against the various
surface molecules.
[0083] As shown in FIG. 2, treatment with HMGN1 (SEQ ID NO: 1)
induces significantly greater expression of costimulatory molecules
(CD80, CD83, and CD 86) and MHC molecules (HLA-ABC and HLA-DR) as
compared to treatment with HMGN2 or without treatment. These
results support the use of HMGN1 (SEQ ID NO: 1) to activate
dendritic cells and enhance an immune response.
Example 4
[0084] This example demonstrates that HMGN1 (SEQ ID NO: 1) enhances
the antigen-presenting capacity of human dendritic cells.
[0085] Human dendritic cells were cultured with RPMI 1640 medium
for 48 hours. Cultures were treated with either HMGN1 (SEQ ID NO:
1) (1 .mu.g/mL) or HMGN2 (1 .mu.g/mL). Culture treated with LPS (1
.mu.g/mL) (as a positive control) and untreated culture (sham)
served as positive and negative controls, respectively. The
cultured cells were then used to stimulate the proliferation of
allogeneic human T cells (10.sup.5) in a mixed lymphocyte reaction.
The proliferation of allogeneic T cells was measured as a function
of tritiated thymidine (.sup.3H-TdR) incorporation. The results are
presented in FIG. 3.
[0086] As shown in FIG. 3, dendritic cells treated with HGMN1
stimulated the proliferation of allogeneic T cells to a degree
equal to or greater than that demonstrated by the positive control.
HMGN2-treated culture and the negative control showed no
significant amount of T-cell proliferation. These results support
the use of HMGN1 (SEQ ID NO: 1) to activate dendritic cells and
enhance an immune response.
Example 5
[0087] This example demonstrates that HMGN1 (SEQ ID NO: 1)
stimulates the maturation of dendritic cells.
[0088] Human dendritic cells were cultured with RPMI 1640 medium
for 48 hours. Cultures were treated with either HMGN1 (SEQ ID NO:
1) (1 .mu.g/mL) or HMGN2 (1 .mu.g/mL). Culture treated with LPS (1
.mu.g/mL) (as a positive control) and untreated culture (sham)
served as positive and negative controls, respectively. The
migratory response of the dendritic cells toward CCL5, CCL21, and
CXCL12 chemokines was measured according to the number of cells per
high power field (No./HPF). The average number of dendritic cells
(mean.+-.SD of triplicate wells) that migrated in response to the
chemokines is presented in FIG. 4.
[0089] As shown in FIG. 4, dendritic cells in the negative control
culture (sham) migrated toward CCL5 and not CCL21, indicating
immaturity. Conversely, dendritic cells cultured in the presence of
HMGN1 (SEQ ID NO: 1) migrated toward CCL21 and not CCL5. These
results indicate that HMGN1 (SEQ ID NO: 1) treatment converted the
dendritic cells from CCL5-responsive to CCL21-responsive, which is
characteristic of dendritic cell maturation. The results support
the use of HMGN1 (SEQ ID NO: 1) to activate dendritic cells and
enhance an immune response.
Example 6
[0090] This example demonstrates that HMGN1 (SEQ ID NO: 1)
stimulates the recruitment of dendritic cells.
[0091] C57BL/6 mice (female, 3/group, 10 weeks old) were injected
intraperitoneally with PBS alone (control) or PBS containing 1
.mu.g of HMGN1 (SEQ ID NO: 1). After 4 hours, the cells in the
peritoneal cavity were washed out, stained with antibodies against
surface markers characteristic of mouse dendritic cells (CD11c+,
CD11c+/CD11b+, CD11c+/B220+, CD11c+/CD11b+/B220+), and analyzed by
flow cytometery. The results are presented in FIG. 5.
[0092] As shown in FIG. 5, HMGN1 (SEQ ID NO: 1) treatment
stimulated the accumulation of various subpopulations of mouse
dendritic cells (CD11c+, CD11c+/CD11b+, CD11c+/B220+,
CD11c+/CD11b+/B220+) into the peritoneal cavity. These results
support the use of HMGN1 (SEQ ID NO: 1) to recruit dendritic cells
and enhance an immune response in vivo.
Example 7
[0093] This example demonstrates that HMGN1 (SEQ ID NO: 1) promotes
an antigen-specific immune response.
[0094] C57BL/6 mice (female, 4/group, 8 weeks old) were
intraperitoneally immunized with ovalbumin (OVA) alone, OVA mixed
with alum (2.5 mg), or HMGN1 (SEQ ID NO: 1) (1 .mu.g) on Day 1,
booster immunized with OVA alone on Day 14, and euthanized on Day
21. The splenocytes of immunized mice were stimulated in vitro with
OVA (0, 2, 10, and 50 .mu.g/ml) for 5 days to measure OVA-specific
proliferation (FIG. 6A) or stimulated with 20 .mu.g/mL of OVA for 3
days (FIG. 6B). Cytokine production was measured, and the results
are presented in FIGS. 6A and 6B.
[0095] As shown in FIG. 6A, the splenocytes of mice immunized with
OVA plus HMGN1 (SEQ ID NO: 1) proliferated vigorously in a dose
dependent manner upon in vitro OVA stimulation, indicating that
HMGN1 (SEQ ID NO: 1) promoted an OVA-specific immune response. As
shown in FIG. 6B, the splenocytes of mice immunized with OVA plus
HMGN1 (SEQ ID NO: 1) produced significant amounts of IFN.gamma. and
TNF.alpha., but not IL-4, indicating that HMGN1 (SEQ ID NO: 1)
predominantly enhanced a Th1-type immune response. Conversely,
alum, which served as a control that predominantly enhances a Th2
immune response, produced a high level of IL-4 and did not increase
IFN.gamma.. These results support the use of HMGN1 (SEQ ID NO: 1)
to enhance an antigen-specific immune response and to shift the
Th-1/Th-2 balance of an immune response of a host towards a Th-1
type immune response.
Example 8
[0096] This example demonstrates the importance of HMGN1 (SEQ ID
NO: 1) to the antigen-specific immune response in vivo.
[0097] HMGN1 knockout (HMGN1-/-) and littermate-matched WT
(HMGN1+/+) mice (4 mice/group) were intra-peritoneally immunized on
day 1 with ovalbumin (OVA) in the presence of alum or LPS, and
boosted on day 14. On day 21, spleens of each group of immunized
mice were pooled for the preparation of single splenocyte
suspension. Subsequently, the splenocytes were stimulated in vitro
with OVA for 48 hours and the cytokine concentrations in the
supernatants were measured by cytokine array.
[0098] As shown in FIGS. 7A and 7B, splenocytes from HMGN1 knockout
mice produced significantly less T cell cytokines (e.g., IL-4,
IL-13, and IFN.gamma.) as compared to wild-type mice, irrespective
of adjuvant used (alum or LPS). These results show that HMGN1 (SEQ
ID NO: 1) plays a significant role in the generation of a T-cell
immune response.
Example 9
[0099] This example demonstrates that the administration of
exogenous HMGN1 (SEQ ID NO: 1) promotes an antigen-specific immune
response in vivo.
[0100] C57BL/6 mice (4/group) were immunized with anthrax vaccine
adsorbed (AVA) alone or in combination with 1-5 .mu.g/mouse of
HMGN1 (SEQ ID NO: 1) on day 1 and day 14 (boost). On day 10 and day
21, serum samples were taken from all mice and anti-AVA specific
antibody titers were measured by ELISA.
[0101] As shown in FIGS. 8A and 8B, mice immunized with AVA+HMGN1
(SEQ ID NO: 1) (1 .mu.g/mouse) produced a higher level of secondary
(day 21) anti-AVA antibodies as compared to mice immunized with AVA
only. HMGN1 (SEQ ID NO: 1) at 5 .mu.g/mouse (5-10 fold) enhanced
both primary (day 10) and secondary (day 21) anti-AVA antibody
responses. These results support the use of HMGN1 (SEQ ID NO: 1) to
enhance an antigen-specific immune response.
Example 10
[0102] This example demonstrates that HMGN1 (SEQ ID NO: 1) induces
dendritic cells to produce cytokines.
[0103] Mice bone marrow-derived DCs were treated with various doses
of HMGN1 (SEQ ID NO: 1) for 24 hours and the production of various
inflammatory cytokines in the supernatants were subsequently
measured by cytokine array.
[0104] As shown in FIGS. 9A and 9B, HMGN1 (SEQ ID NO: 1) stimulated
the production of various inflammatory cytokines (TNF.alpha.,
IL-1.beta., keratinocyte chemoattractant (KC), IL-10, IL-6, and
IL-12p70) by mouse DCs. The results support the use of HMGN1 (SEQ
ID NO: 1) to activate and/or recruit dendritic cells and modulate
an immune response.
Example 11
[0105] This example demonstrates to importance of HMGN1 (SEQ ID NO:
1) to the inflammatory immune response in vivo.
[0106] HMGN1 WT (HMGN1+/+) or KO (HMGN1-/-) mice (4 mice/group)
were immunized intraperitoneally with OVA alone or OVA in the
presence of alum or LPS (endotoxin). At 24 or 96 hours after the
immunization, mouse serum samples were taken and various cytokines
were measured.
[0107] As shown in FIG. 10A, HMGN1 KO mice failed to produce
detectable levels of the inflammatory cytokines tested (IL-1.beta.,
IL-2, IL-6, and IL-12p70) at 24 and 96 hours after immunization,
and produced less TNF.alpha. at 96 hours as compared to HMGN1 WT
mice, irrespective of the adjuvant used for the immunization
(either alum or LPS). These results demonstrate the importance of
HMGN1 (SEQ ID NO: 1) in the induction of inflammatory cytokines,
and support the use of HMGN1 (SEQ ID NO: 1) as a basis to modulate
the inflammatory response, such as through the use of HMGN1 (SEQ ID
NO: 1) inhibitory molecules.
Example 12
[0108] This example demonstrates that HMGN1 (SEQ ID NO: 1)
stimulates the activation of dendritic cells.
[0109] Mouse bone marrow-derived DCs were untreated or treated with
HMGN1 (SEQ ID NO: 1) (at 1 .mu.g/ml) at 37.degree. C. for 20 or 60
minutes. At the end of treatment, DCs were washed extensively with
ice-cold PBS, pelleted, and solubilized in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) lysis buffer (at
10.sup.7/ml) to make cell lysate. DC lysates were then loaded onto
SDS-PAGE gels, separated by electrophoresis, and transferred onto
pieces of polyvinylidene fluoride (PVDF) membrane.
[0110] The PVDF membranes were analyzed by Western blot. Briefly,
the membranes were washed, blocked, and reacted with rabbit
anti-I-.kappa.B.alpha., anti-phosphorylated p44/42
mitogen-activated protein kinases (MAPKs), anti-phosphorylated p38
MAPK, or anti-phosphorylated c-Jun N-terminal kinase (JNK) MAPK
antibodies in a cold room overnight. After removal of unbound
antibodies by washes, the PVDF membranes were reacted with
horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody,
washed, developed with an Amersham enhanced luminol-based
chemiluminescent (ECL.TM.) kit, and autoradiographed. The PVDF
membranes were then stripped and re-probed with
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-p44/42,
anti-p38, and anti-JNK antibodies, respectively.
[0111] The results are summarized in FIG. 11. As shown in FIG. 11,
HMGN1 (SEQ ID NO: 1) treatment of mouse DCs decreased the level of
I-.kappa.B.alpha. by the 60 minute time point, indicating the
activation of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-.kappa.B) in DCs. Similar band intensity for
the three lanes confirms that a similar amount of total DC lysate
proteins was loaded into each lane.
[0112] For the MAPKs, HMGN1 (SEQ ID NO: 1) treatment caused
phosphorylation of three classes of MAPKs in a time-dependent
manner (as evidenced by the intensified bands of phosphorylated
p44/42, phosphorylated p38, and phosphorylated JNK), indicating the
activation of three classes of MAPKs. The bands of p44/42, p38, and
JNK were similar between 0, 20, and 60 minute time points,
indicating that HMGN1 (SEQ ID NO: 1) treatment did not change the
level of unactivated MAPKs.
[0113] These results support the use of HMGN1 (SEQ ID NO: 1) to
activate dendritic cells and enhance an immune response.
[0114] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0115] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0116] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
141100PRTHomo sapiens 1Met Pro Lys Arg Lys Val Ser Ser Ala Glu Gly
Ala Ala Lys Glu Glu 1 5 10 15 Pro Lys Arg Arg Ser Ala Arg Leu Ser
Ala Lys Pro Pro Ala Lys Val 20 25 30 Glu Ala Lys Pro Lys Lys Ala
Ala Ala Lys Asp Lys Ser Ser Asp Lys 35 40 45 Lys Val Gln Thr Lys
Gly Lys Arg Gly Ala Lys Gly Lys Gln Ala Glu 50 55 60 Val Ala Asn
Gln Glu Thr Lys Glu Asp Leu Pro Ala Glu Asn Gly Glu 65 70 75 80 Thr
Lys Thr Glu Glu Ser Pro Ala Ser Asp Glu Ala Gly Glu Lys Glu 85 90
95 Ala Lys Ser Asp 100 299PRTHomo sapiens 2Met Pro Lys Arg Lys Ser
Pro Glu Asn Thr Glu Gly Lys Asp Gly Ser 1 5 10 15 Lys Val Thr Lys
Gln Glu Pro Thr Arg Arg Ser Ala Arg Leu Ser Ala 20 25 30 Lys Pro
Ala Pro Pro Lys Pro Glu Pro Lys Pro Arg Lys Thr Ser Ala 35 40 45
Lys Lys Glu Pro Gly Ala Lys Ile Ser Arg Gly Ala Lys Gly Lys Lys 50
55 60 Glu Glu Lys Gln Glu Ala Gly Lys Glu Gly Thr Ala Pro Ser Glu
Asn 65 70 75 80 Gly Glu Thr Lys Ala Glu Glu Ala Gln Lys Thr Glu Ser
Val Asp Asn 85 90 95 Glu Gly Glu 377PRTHomo sapiens 3Met Pro Lys
Arg Lys Ser Pro Glu Asn Thr Glu Gly Lys Asp Gly Ser 1 5 10 15 Lys
Val Thr Lys Gln Glu Pro Thr Arg Arg Ser Ala Arg Leu Ser Ala 20 25
30 Lys Pro Ala Pro Pro Lys Pro Glu Pro Lys Pro Arg Lys Thr Ser Ala
35 40 45 Lys Lys Glu Pro Gly Ala Lys Ile Ser Arg Gly Ala Lys Gly
Lys Lys 50 55 60 Glu Glu Lys Gln Glu Ala Gly Lys Glu Gly Thr Glu
Asn 65 70 75 490PRTHomo sapiens 4Met Pro Lys Arg Lys Ala Lys Gly
Asp Ala Lys Gly Asp Lys Ala Lys 1 5 10 15 Val Lys Asp Glu Pro Gln
Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro 20 25 30 Ala Pro Pro Lys
Pro Glu Pro Arg Pro Lys Lys Ala Ser Ala Lys Lys 35 40 45 Gly Glu
Lys Leu Pro Lys Gly Arg Lys Gly Lys Ala Asp Ala Gly Lys 50 55 60
Asp Gly Asn Asn Pro Ala Lys Asn Arg Asp Ala Ser Thr Leu Gln Ser 65
70 75 80 Gln Lys Ala Glu Gly Thr Gly Asp Ala Lys 85 90 5282PRTHomo
sapiens 5Met Pro Lys Arg Lys Ala Ala Gly Gln Gly Asp Met Arg Gln
Glu Pro 1 5 10 15 Lys Arg Arg Ser Ala Arg Leu Ser Ala Met Leu Val
Pro Val Thr Pro 20 25 30 Glu Val Lys Pro Lys Arg Thr Ser Ser Ser
Arg Lys Met Lys Thr Lys 35 40 45 Ser Asp Met Met Glu Glu Asn Ile
Asp Thr Ser Ala Gln Ala Val Ala 50 55 60 Glu Thr Lys Gln Glu Ala
Val Val Glu Glu Asp Tyr Asn Glu Asn Ala 65 70 75 80 Lys Asn Gly Glu
Ala Lys Ile Thr Glu Ala Pro Ala Ser Glu Lys Glu 85 90 95 Ile Val
Glu Val Lys Glu Glu Asn Ile Glu Asp Ala Thr Glu Lys Gly 100 105 110
Gly Glu Lys Lys Glu Ala Val Ala Ala Glu Val Lys Asn Glu Glu Glu 115
120 125 Asp Gln Lys Glu Asp Glu Glu Asp Gln Asn Glu Glu Lys Gly Glu
Ala 130 135 140 Gly Lys Glu Asp Lys Asp Glu Lys Gly Glu Glu Asp Gly
Lys Glu Asp 145 150 155 160 Lys Asn Gly Asn Glu Lys Gly Glu Asp Ala
Lys Glu Lys Glu Asp Gly 165 170 175 Lys Lys Gly Glu Asp Gly Lys Gly
Asn Gly Glu Asp Gly Lys Glu Lys 180 185 190 Gly Glu Asp Glu Lys Glu
Glu Glu Asp Arg Lys Glu Thr Gly Asp Gly 195 200 205 Lys Glu Asn Glu
Asp Gly Lys Glu Lys Gly Asp Lys Lys Glu Gly Lys 210 215 220 Asp Val
Lys Val Lys Glu Asp Glu Lys Glu Arg Glu Asp Gly Lys Glu 225 230 235
240 Asp Glu Gly Gly Asn Glu Glu Glu Ala Gly Lys Glu Lys Glu Asp Leu
245 250 255 Lys Glu Glu Glu Glu Gly Lys Glu Glu Asp Glu Ile Lys Glu
Asp Asp 260 265 270 Gly Lys Lys Glu Glu Pro Gln Ser Ile Val 275 280
6300DNAHomo sapiens 6atgcccaaga ggaaggtcag ctccgccgaa ggcgccgcca
aggaagagcc caagaggaga 60tcggcgcggt tgtcagctaa acctcctgca aaagtggaag
cgaagccgaa aaaggcagca 120gcgaaggata aatcttcaga caaaaaagtg
caaacaaaag ggaaaagggg agcaaaggga 180aaacaggccg aagtggctaa
ccaagaaact aaagaagact tacctgcgga aaacggggaa 240acgaagactg
aggagagtcc agcctctgat gaagcaggag agaaagaagc caagtctgat
3007297DNAHomo sapiens 7atgccgaaga gaaagtctcc agagaataca gagggcaaag
atggatccaa agtaactaaa 60caggagccca caagacggtc tgccagattg tcagcgaaac
ctgctccacc aaaacctgaa 120cccaaaccaa gaaaaacatc tgctaagaaa
gaacctggag caaagattag cagaggtgct 180aaagggaaga aggaggaaaa
gcaggaagct ggaaaggaag gtactgcacc atctgaaaat 240ggtgaaacta
aagctgaaga ggcacagaaa actgaatctg tagataacga gggagaa 2978231DNAHomo
sapiens 8atgccgaaga gaaagtctcc agagaataca gagggcaaag atggatccaa
agtaactaaa 60caggagccca caagacggtc tgccagattg tcagcgaaac ctgctccacc
aaaacctgaa 120cccaaaccaa gaaaaacatc tgctaagaaa gaacctggag
caaagattag cagaggtgct 180aaagggaaga aggaggaaaa gcaggaagct
ggaaaggaag gcacagaaaa c 2319270DNAHomo sapiens 9atgcccaaga
gaaaggcaaa aggagatgct aaaggtgata aagcaaaggt gaaggatgag 60ccacagagga
gatcagctcg gttgtctgct aaaccagctc ctccaaaacc agagcccagg
120cctaaaaagg cctctgcaaa gaagggagag aagcttccca aagggagaaa
ggggaaagca 180gatgctggaa aggatgggaa caaccctgca aaaaaccgag
atgcctctac actccagtcc 240cagaaagcgg aaggcactgg ggatgccaag
27010846DNAHomo sapiens 10atgcccaaaa gaaaggctgc aggtcaaggt
gatatgaggc aggagccaaa gagaagatct 60gccaggttgt ctgctatgct tgtgccagtt
acaccagagg tgaagcctaa aagaacatca 120agttcaagga aaatgaagac
aaaaagtgat atgatggaag aaaacataga tacaagtgcc 180caagcagttg
ctgaaaccaa gcaagaagca gttgttgaag aagactacaa tgaaaatgct
240aaaaatggag aagccaaaat tacagaggca ccagcttctg aaaaagaaat
tgtggaagta 300aaagaagaaa atattgaaga tgccacagaa aagggaggag
aaaagaaaga agcagtggca 360gcagaagtaa aaaatgaaga agaagatcag
aaagaagatg aagaagatca aaacgaagag 420aaaggggaag ctggaaaaga
agacaaagat gaaaaagggg aagaagatgg aaaagaggat 480aaaaatggaa
atgagaaagg agaagatgca aaagagaaag aagatggaaa aaaaggtgaa
540gacggaaaag gaaatggaga agatggaaaa gagaaaggag aagatgaaaa
agaggaagaa 600gacagaaaag aaacaggaga tggaaaagag aatgaagatg
gaaaagagaa gggagataaa 660aaagagggga aagatgtaaa agtcaaagaa
gatgaaaaag agagagaaga tggaaaagaa 720gatgaaggtg gaaatgagga
agaagctgga aaagagaaag aagatttaaa agaagaggaa 780gaaggaaaag
aggaagatga gatcaaagaa gatgatggaa aaaaagagga gccacagagt 840attgtt
8461136PRTHomo sapiens 11Lys Glu Glu Pro Lys Arg Arg Ser Ala Arg
Leu Ser Ala Lys Pro Pro 1 5 10 15 Ala Lys Val Glu Ala Lys Pro Lys
Lys Ala Ala Ala Lys Asp Lys Ser 20 25 30 Ser Asp Lys Lys 35
1230PRTHomo sapiens 12Lys Gln Glu Pro Thr Arg Arg Ser Ala Arg Leu
Ser Ala Lys Pro Ala 1 5 10 15 Pro Pro Lys Pro Glu Pro Lys Pro Arg
Lys Thr Ser Ala Lys 20 25 30 1330PRTHomo sapiens 13Lys Asp Glu Pro
Gln Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro Ala 1 5 10 15 Pro Pro
Lys Pro Glu Pro Arg Pro Lys Lys Ala Ser Ala Lys 20 25 30
1430PRTHomo sapiens 14Lys Arg Arg Ser Ala Arg Leu Ser Ala Met Leu
Val Pro Val Thr Pro 1 5 10 15 Glu Val Lys Pro Lys Arg Thr Ser Ser
Ser Arg Lys Met Lys 20 25 30
* * * * *