U.S. patent application number 16/713202 was filed with the patent office on 2020-11-26 for peptide for inducing regeneration of tissue and use thereof.
The applicant listed for this patent is OSAKA UNIVERSITY, STEMRIM INC.. Invention is credited to MAYUMI ENDO, NATSUMI HAMABUCHI, TSUTOMU KANEZAKI, KANA NAITO, YUKIKO NOGUCHI, SHIGERU SAKURAI, KATSUTO TAMAI, TAKEHIKO YAMAZAKI.
Application Number | 20200369736 16/713202 |
Document ID | / |
Family ID | 1000005004285 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200369736 |
Kind Code |
A1 |
TAMAI; KATSUTO ; et
al. |
November 26, 2020 |
PEPTIDE FOR INDUCING REGENERATION OF TISSUE AND USE THEREOF
Abstract
(Objective) An objective of the present invention is to provide
therapeutic agents that, in association with stimulation of
PDGFR.alpha.-positive cells such as bone marrow mesenchymal stem
cells, promote their mobilization into blood and accumulation in a
damaged tissue, and induce tissue regeneration in a living body.
(Means for solution) Multiple peptides were synthesized, and the
migration-promoting activity of each peptide was evaluated. As a
result, the present inventors successfully identified multiple
peptides that have migration-promoting activity on a
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line
(MSC-1). Further, the present inventors confirmed that the
identified peptides also have migration-promoting activity on skin
fibroblasts, which are PDGFR.alpha.-positive cells.
Inventors: |
TAMAI; KATSUTO; (OSAKA,
JP) ; YAMAZAKI; TAKEHIKO; (OSAKA, US) ;
KANEZAKI; TSUTOMU; (OSAKA, JP) ; SAKURAI;
SHIGERU; (OSAKA, JP) ; NOGUCHI; YUKIKO;
(OSAKA, JP) ; ENDO; MAYUMI; (OSAKA, JP) ;
HAMABUCHI; NATSUMI; (OSAKA, JP) ; NAITO; KANA;
(OSAKA, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STEMRIM INC.
OSAKA UNIVERSITY |
OSAKA
OSAKA |
|
JP
JP |
|
|
Family ID: |
1000005004285 |
Appl. No.: |
16/713202 |
Filed: |
December 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15691017 |
Aug 30, 2017 |
10550165 |
|
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16713202 |
|
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14114395 |
Feb 6, 2014 |
10364276 |
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PCT/JP2012/059113 |
Apr 3, 2012 |
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15691017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4705 20130101;
A61K 38/17 20130101; C07K 14/4702 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 26, 2011 |
JP |
2011-098270 |
Oct 3, 2011 |
JP |
2011-219454 |
Claims
1. A peptide comprising the amino acid sequence of position 17 to
position 25 in the amino acid sequence of SEQ ID NO: 1 and having
an activity of stimulating migration of a cell, wherein the peptide
consists of 195 amino acids or less.
2. The peptide of claim 1, which is a peptide consisting of 185
amino acids or less.
3. The peptide of claim 1, which is a peptide consisting of 84
amino acids or less.
4. The peptide of claim 1, which is a peptide consisting of 44
amino acids or less.
5. The peptide of claim 1, which comprises the amino acid sequences
of position 13 to position 25 in the amino acid sequence of SEQ ID
NO: 1.
6. The peptide of claim 1, which comprises the amino acid sequences
of position 10 to position 25 in the amino acid sequence of SEQ ID
NO: 1.
7. The peptide of claim 1, which comprises the amino acid sequences
of position 11 to position 44 in the amino acid sequence of SEQ ID
NO: 1.
8. The peptide of claim 1, which comprises the amino acid sequences
of position 11 to position 34 in the amino acid sequence of SEQ ID
NO: 1.
9. The peptide of claim 1, which comprises the amino acid sequences
of position 17 to position 30 in the amino acid sequence of SEQ ID
NO: 1.
10. The peptide of claim 1, which comprises the amino acid
sequences of position 14 to position 30 in the amino acid sequence
of SEQ ID NO: 1.
11. The peptide of claim 1, which comprises the amino acid
sequences of position 11 to position 30 in the amino acid sequence
of SEQ ID NO: 1.
12. The peptide of claim 1, which is a synthetic peptide.
13. The peptide of claim 1, which is a peptide produced using a
cell.
14. The peptide of claim 1, which is a peptide to which a tag is
added.
15. The peptide of claim 1, which is a peptide to which a
tag-derived peptide fragment is added.
16. A composition comprising the peptide of claim 1 and a
pharmaceutically acceptable carrier or additive.
17. The composition of claim 16, wherein the pharmaceutically
acceptable carrier or additive is selected from the group
consisting of surfactant, excipient, colorant, perfume,
preservative, stabilizer, buffer, suspending agent, isotonizing
agent, binder, disintegrant, lubricant, flow promoter, and
flavoring agent.
18. The composition of claim 16, wherein the pharmaceutically
acceptable carrier or additive is selected from the group
consisting of light anhydrous silicic acid, lactose, crystalline
cellulose, mannitol, starch, carmellose calcium, carmellose sodium,
hydroxypropylcellulose, hydroxypropylmethylcellulose,
polyvinylacetaldiethylamino acetate, polyvinylpyrrolidone, gelatin,
medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated
castor oil 60, white sugar, carboxymethyl cellulose, corn starch,
and inorganic salt.
19. A DNA encoding the peptide of claim 1.
20. A vector comprising the DNA of claim 19.
21. A transformed cell comprising the DNA of claim 19.
22. A transformed cell comprising the vector of claim 20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application Ser. No. 15/691,017, filed Aug. 30, 2017; which is a
continuation application of application Ser. No. 14/114,395, filed
Feb. 6, 2014, now U.S. Pat. No. 10,364,276; which is a National
Stage Application of International Application Number
PCT/JP2012/059113, filed Apr. 3, 2012; which claims priority to
Japanese Application No. 2011-098270, filed Apr. 26, 2011; and
Japanese Application No. 2011-219454, filed Oct. 3, 2011; all of
which are incorporated herein by reference in their entirety.
[0002] The Sequence Listing for this application is labeled
"SeqList-18Feb14.txt", which was created on Feb. 18, 2014, and is
13 KB. The entire content is incorporated herein by reference in
its entirety.
TECHNICAL FIELD
[0003] The present invention relates to peptides for inducing
tissue regeneration and uses thereof.
BACKGROUND OF THE INVENTION
[0004] It has been becoming clear that each organ or tissue in the
living organism has tissue stem cells that maintain its structural
and functional homeostasis. For example, cardiac stem cells are
present in the heart, neural stem cells are present in the brain,
and epidermal stem cells and hair follicle stem cells are present
in the skin. They provide cardiomyocytes, neurons, and epidermal
cells and hair follicle epithelial cells to the heart, brain, and
skin, respectively, over a lifetime to maintain their structures
and functions. Meanwhile, hematopoietic stem cells, which
differentiate into blood cells such as erythrocytes, leukocytes,
and platelets, are present in the bone marrow. The blood cells
derived from hematopoietic stem cells circulate through all organs
or tissues in the body via blood flow and serve essential functions
for the maintenance of life, such as oxygen supply, immune
response, arrest of hemorrhage, and repair of damaged tissues.
Thus, it is fair to say that bone-marrow hematopoietic stern cells
contribute to maintaining the homeostasis of all tissues in the
body via peripheral circulation, rather than maintaining the
homeostasis of bone marrow and bone tissues where they are
localized.
[0005] Recently, it has been demonstrated that, in addition to
hematopoietic stem cells, mesenchymal stem cells capable of
differentiating into not only mesodermal tissues such as bone,
cartilage, and adipose but also ectodermal tissues such as neuron
and epidermis are present in the bone marrow. However, little is
understood about the significance of the presence of mesenchymal
stem cells in the living body. However, given that hematopoietic
stem cells that maintain the homeostasis of all organs and tissues
by supplying blood cells via peripheral circulation are present in
the bone marrow, it is expected that mesenchymal stem cells present
in the bone marrow may also contribute to the homeostatic
maintenance of living tissues by supplying cells capable of
differentiating into bone, cartilage, adipose, neuron, epithelium,
etc., to tissues or organs in need thereof in the living body via
peripheral circulation.
[0006] Currently, regenerative medicine is under intensive
development, in which bone marrow mesenchymal stem cells are
prepared by collecting bone-marrow blood, and after expansion by
cell culture, the cells are grafted into the site of intractable
tissue damage or into peripheral circulation to induce regeneration
of the damaged tissue. Clinical application of bone marrow
mesenchymal stem cell transplantation has already been underway in
regenerative medicine for cerebral infarction, cardiac infarction,
intractable skin ulcer, etc. Furthermore, transplanted bone marrow
mesenchymal stem cells have been demonstrated to produce the effect
of suppressing inflammation and immune response as well as the
effect of suppressing fibrous scar formation at local sites in the
body. Clinical trials have begun on bone marrow mesenchymal stem
cell transplantation therapy as a new therapeutic method to treat
scleroderma, which is an autoimmune disease, or to treat graft
versus host disease (GVHD), which is a serious side effect after
bone marrow transplantation or blood infusion. However, bone-marrow
blood containing bone marrow mesenchymal stem cells is collected
only by an invasive method where thick needles are repeatedly
inserted into the iliac bone. In addition, continuous passages of
bone marrow mesenchymal stem cells outside the body lead to gradual
loss of their proliferative ability and multipotency. Moreover,
since culturing bone marrow mesenchymal stem cells with high
quality control for ensuring the safety of in vivo transplantation
requires special cell culture facilities such as cell processing
center (CPC), it can only be performed currently in very limited
universities and companies. Thus, in order to make the regenerative
medicine using bone marrow mesenchymal stem cells available to a
large number of patients around the world suffering from
intractable tissue damage, it is an urgent task to develop
techniques for mesenchymal stem cell regenerative medicine that can
be performed in any medical facilities.
[0007] High mobility group box 1 (HMGB1) protein was identified
about 30 years ago as a non-histone chromatin protein that
regulates gene expression and DNA repair by regulating the
structure of nuclear chromatin. The structure of the HMGB1 protein
is primarily constituted by two DNA-binding domains, and those at
the N- and C-terminal are referred to as A-box and B-box,
respectively. Past studies have revealed that the domain which
binds TLR to induce inflammatory reaction is located within the
B-box of the HMGB1 molecule.
PRIOR-ART DOCUMENTS
Patent Documents
[0008] Patent Document 1: WO2008/053892 [0009] Patent Document 2:
WO2007/015546 [0010] Patent Document 3: WO2009/133939 [0011] Patent
Document 4: WO2009/133943 [0012] Patent Document 5: WO2009/133940
[0013] Patent Document 6: Japanese Patent Kohyo Publication No.
(JP-A) 2005-537253 (unexamined Japanese national phase publication
corresponding to a non-Japanese international publication)
Non-Patent Documents
[0013] [0014] Non-patent Document 1: Bustin et al., Mol Cell Biol,
19: 5237-5246, 1999 [0015] Non-patent Document 2: Hori et al., J.
Biol. Chem., 270, 25752-25761, 1995 [0016] Non-patent Document 3:
Wang et al., Science, 285: 248-251, 1999 [0017] Non-patent Document
4: Muller et al., EMBO J, 20: 4337-4340, 2001 [0018] Non-patent
Document 5: Wang et al., Science, 285: 248-251, 1999 [0019]
Non-patent Document 6: Germani et al., J Leukoc Biol. January;
81(1): 41-5, 2007 [0020] Non-patent Document 7: Palumbo et al., J.
Cell Biol., 164: 441-449, 2004 [0021] Non-patent Document 8:
Merenmies et al., J. Biol. Chem., 266: 16722-16729, 1991 [0022]
Non-patent Document 9: Wu Y et al., Stem cells, 25: 2648-2659, 2007
[0023] Non-patent Document 10: Tamai et al., Proc Natl Acad Sci
USA. 2011 Apr. 4. [Epub ahead of print], 108: 6609-6614, 2011
[0024] Non-patent Document 11: Yang et al., J Leukoc Biol. January;
81(1): 59-66, 2007
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0025] The present inventors recently conducted studies to
elucidate the mechanism of regeneration of exfoliated epidermis in
"epidermolysis bullosa", which is an intractable hereditary skin
disorder showing skin exfoliation of the whole body and burn-like
symptoms all over the body due to abnormality in the gene of an
adhesion molecule in the basal membrane region of skin. Using
epidermolysis bullosa model mice transplanted with green
fluorescent protein (GFP) transgenic bone marrow cells, the present
inventors revealed that the high mobility group box 1 (HMGB1)
protein released from exfoliated epidermis to blood stimulates and
recruits platelet-derived growth factor receptor alpha
(PDGFR.alpha.)-positive cells from bone marrow to blood and thereby
promotes the accumulation of the cells to the site of epidermal
exfoliation, and that PDGFR.alpha.-positive cells localized at the
site of epidermal exfoliation differentiated into fibroblasts and
epidermal cells and had a great contribution to the regeneration of
damaged skin. The present inventors also revealed that, when a
recombinant HMGB1 protein was administered via the caudal vein
after induction of skin ulcer or cerebral infarction in mice,
PDGFR.alpha.-positive cells were recruited from the bone marrow
into the blood and accumulated to the site of skin ulceration or
cerebral infarction, thereby strongly inducing regeneration from
skin ulceration or cerebral infarction. Intramedullary
PDGFR.alpha.-positive cells have been previously reported to be
mesenchymal stem cells capable of differentiating into bone,
cartilage, and adipose, and also into neuron and epithelium. Thus,
it has been found possible to allow many mesenchymal stem cells to
accumulate at damaged tissues in the living organism by
administering HMGB1 to mobilize intramedullary
PDGFR.alpha.-positive mesenchymal stem cells into peripheral
circulation, without performing special ex vivo culture of cells
collected from the body.
[0026] If HMGB1 is developed into a pharmaceutical agent for
inducing regeneration of damaged tissues by recruiting bone marrow
mesenchymal stem cells to blood in the body, every medical facility
will be able to perform regenerative medicine based on bone marrow
mesenchymal stem cells. This will solve many problems that the
above-mentioned current bone marrow mesenchymal stem cell-based
regenerative medicine faces.
[0027] As described above, HMGB1 pharmaceuticals are revolutionary
therapeutic agents that promote the recruitment of bone marrow
mesenchymal stem cells into blood and accumulation of the cells to
damaged tissues, thereby inducing tissue regeneration in the body.
In previous studies conducted by the present inventors, no side
effects were observed even when a high concentration of recombinant
HMGB1 protein was administered to mice or rats. In view of this, as
well as the fact observed by the present inventors that a
significantly high level of HMGB1 is present in the peripheral
blood of epidermolysis bullosa patients who have no severe symptoms
except epidermal exfoliation, it is expected that HMGB1
administration is highly safe. However, there are also reports that
HMGB1 has an inflammatory effect. As described above, there are
several findings on HMGB1; however, nothing is known about the
effect of fragments of the HMGB1 protein on mesenchymal stem cells
or what roles they play in tissue regeneration.
Means for Solving the Problems
[0028] The present inventors had a peptide consisting of amino
acids at positions 1 to 84 of an HMGB1 protein and a peptide
consisting of amino acids at positions 85 to 169 of the HMGB1
protein respectively secreted as recombinant proteins into HEK293
cell media. The proteins of interest in the media were each
purified by chromatography, and their migration-promoting activity
on the PDGFR.alpha.-positive bone marrow mesenchymal stem cell line
(MSC-1) was examined. As a result, the present inventors found that
the peptide consisting of amino acids of positions 1 to 84 showed
migration-promoting activity.
[0029] Then, based on the peptide consisting of amino acids at
positions 1 to 84 of the HMGB1 protein which was confirmed to have
migration-promoting activity on MSC-1, the present inventors
prepared a peptide consisting of amino acids at positions 1 to 44
and a peptide consisting of amino acids at positions 45 to 84, and
examined each peptide for migration-promoting activity. The results
showed that both peptide fragments exhibited migration-promoting
activity on the PDGFR.alpha.-positive bone marrow mesenchymal stem
cell line (MSC-1).
[0030] Then, various peptide fragments overlapping each other
around the above respective fragments were chemically synthesized,
and evaluated for their migration-promoting activity on the
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line
(MSC-1). As a result, the present inventors identified several
peptides showing migration-promoting activity.
[0031] Further, the present inventors confirmed that the identified
peptides have migration-promoting activity on skin fibroblasts,
which are PDGFR.alpha.-positive, and have an effect of reducing the
size of cerebral infarcts in a cerebral infarction model mouse.
[0032] The present inventors had a recombinant protein consisting
of amino acids of positions 2 to 84 of the HMGB1 protein and a
recombinant protein consisting of amino acids of positions 89 to
215 of the HMGB1 protein expressed in E. coli. The expressed
proteins were purified by column chromatography, and examined for
their migration-promoting activity on the PDGFR.alpha.-positive
bone marrow mesenchymal stem cell line (MSC-1) and on human bone
marrow mesenchymal stem cells. As a result, the present inventors
confirmed migration-promoting activity of the peptide consisting of
amino acids of positions 2 to 84 and the peptide consisting of
amino acids of positions 89 to 215.
[0033] Next, based on the peptide consisting of amino acids of
positions 2 to 84 which was confirmed to have migration-promoting
activity on MSC-1 and human bone marrow mesenchymal stern cells,
the present inventors prepared a peptide consisting of amino acids
of positions 2 to 44 and a peptide consisting of amino acids of
positions 45 to 84, and examined them for their migration-promoting
activities. The result showed that both peptide fragments prepared
exhibited migration-promoting activity on MSC-1 and human bone
marrow mesenchymal stem cells.
[0034] Next, based on the peptide consisting of amino acids of
positions 89 to 215 which was confirmed to have migration-promoting
activity on MSC-1 and human bone marrow mesenchymal stem cells, the
present inventors prepared peptides with the C-terminus
increasingly shortened, i.e. a peptide consisting of amino acids of
positions 89 to 205, a peptide consisting of amino acids of
positions 89 to 195, and a peptide consisting of amino acids of
positions 89 to 185, and examined each of them for
migration-promoting activity. The result showed that among the
prepared peptide fragments, those with a shorter C terminus
demonstrated more enhanced migration-promoting activity on the
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line
(MSC-1) and human bone marrow mesenchymal stem cells.
[0035] Further, when three types of fusion peptides were generated
by adding to the peptide consisting of amino acids of positions 2
to 84, a whole or partial C-terminal acidic tail consisting of
aspartic acid and glutamic acid (10-, 20-, or 30-amino acid
sequence), it was surprisingly found that the migration-promoting
activity of the 2-84 peptide was extremely reduced for all fusion
peptides. This shows that a whole or partial acidic tail
suppressively regulates the migration-promoting activity of
full-length HMGB 1. It became clear by the above fragmentation that
there are at least three or more migration-promoting activity
domains, and this suggests that these domains in their full-length
state might be suppressed by the acidic tail.
[0036] Further, the inventors confirmed that the identified
peptides have therapeutic effect in a damaged skin model.
[0037] Based on these findings, the present application provides
the following: [0038] [1] a composition for use in stimulating
migration of a cell, comprising a substance of any of (a) to (c)
below:
[0039] (a) a peptide consisting of a portion of an HMGB1 protein
and having an activity of stimulating migration of a cell;
[0040] (b) a cell secreting the peptide of (a); and
[0041] (c) a vector into which a DNA encoding the peptide of (a) is
inserted; [0042] [2] A composition for use in mobilizing a cell
from bone marrow to peripheral blood, comprising a substance of (a)
to (c) below: [0043] (a) a peptide consisting of a portion of an
HMGB1 protein and having an activity of stimulating migration of a
cell; [0044] (b) a cell secreting the peptide of (a); and [0045]
(c) a vector into which a DNA encoding the peptide of (a) is
inserted; [0046] [3] a composition for use in regenerating a
tissue, comprising a substance of any of (a) to (c) below: [0047]
(a) a peptide consisting of a portion of an HMGB1 protein and
having an activity of stimulating migration of a cell; [0048] (b) a
cell secreting the peptide of (a); and [0049] (c) a vector into
which a DNA encoding the peptide described in (a) is inserted;
[0050] [4] the composition of any one of embodiments 1-3, wherein
the cell stimulated to migrate or mobilized from bone marrow to
peripheral blood is a PDGFR.alpha.-positive cell: [0051] [5] the
composition of any one of embodiments 1-4, wherein the cell
stimulated to migrate or mobilized from bone marrow to peripheral
blood is a stem cell; [0052] [6] the composition of any one of
embodiments 1-5, wherein the cell stimulated to migrate or
mobilized from bone marrow to peripheral blood is a bone marrow
cell; [0053] [7] the composition of any one of embodiments 1-6,
wherein the cell stimulated to migrate or mobilized from bone
marrow to peripheral blood is a bone marrow mesenchymal stem cell;
[0054] [8] the composition of any one of embodiments 1-7, wherein
the peptide consisting of a portion of an HMGB1 protein and having
an activity of stimulating migration of a cell is a peptide
consisting of the whole or part of the amino acid sequence of
positions 1 to 195 or positions 1 to 185 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5, and having an activity of
stimulating migration of a cell; [0055] [9] the composition of any
one of embodiments 1-7, wherein the peptide consisting of a portion
of an HMGB1 protein and having an activity of stimulating migration
of a cell is a peptide comprising any of the amino acid sequences
below and having an activity of stimulating migration of a cell:
[0056] (1) the amino acid sequence of position 17 to position 25 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5;
[0057] (2) the amino acid sequence of position 45 to position 74 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5;
[0058] (3) the amino acid sequence of position 55 to position 84 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5;
[0059] (4) the amino acid sequence of position 85 to position 169
in the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5;
and [0060] (5) the amino acid sequence of position 89 to position
185 in the amino acid sequence of any one of SEQ ID NOs: 1, 3, and
5; [0061] [10] the composition of any one of embodiments 1-7,
wherein the peptide consisting of a portion of an HMGB1 protein and
having an activity of stimulating migration of a cell is a peptide
having an activity of stimulating migration of a cell which
consists of the whole or part of the amino acid sequence of
positions 1 to 195 or positions 1 to 185 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5, and comprises any of the
amino acid sequences below: [0062] (1) the amino acid sequence of
position 17 to position 25 in the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5; [0063] (2) the amino acid sequence of
position 45 to position 74 in the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5; [0064] (3) the amino acid sequence of
position 55 to position 84 in the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5; [0065] (4) the amino acid sequence of
position 85 to position 169 in the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5; and [0066] (5) the amino acid sequence
of position 89 to position 185 in the amino acid sequence of any
one of SEQ ID NOs: 1, 3, and 5; [0067] [11] a composition for use
in stimulating migration of a cell, which comprises a peptide
comprising any of the amino acid sequences below and having an
activity of stimulating migration of a cell: [0068] (1) the amino
acid sequence of position 17 to position 25 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0069] (2) the
amino acid sequence of position 45 to position 74 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0070] (3) the
amino acid sequence of position 55 to position 84 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0071] (4) the
amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; and [0072] (5)
the amino acid sequence of position 89 to position 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; [0073] [12] a
composition for use in mobilizing a cell from bone marrow to
peripheral blood, which comprises a peptide comprising any of the
amino acid sequences below and having an activity of stimulating
migration of a cell: [0074] (1) the amino acid sequence of position
17 to position 25 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5; [0075] (2) the amino acid sequence of position 45
to position 74 in the amino acid sequence of any one of SEQ ID NOs:
1, 3, and 5; [0076] (3) the amino acid sequence of position 55 to
position 84 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5; [0077] (4) the amino acid sequence of position 85 to
position 169 in the amino acid sequence of any one of SEQ ID NOs:
1, 3, and 5; and [0078] (5) the amino acid sequence of position 89
to position 185 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5; [0079] [13] a composition for in regenerating a
tissue, which comprises a peptide comprising any of the amino acid
sequences below and having an activity of stimulating migration of
a cell: [0080] (1) the amino acid sequence of position 17 to
position 25 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5; [0081] (2) the amino acid sequence of position 45 to
position 74 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5; [0082] (3) the amino acid sequence of position 55 to
position 84 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5; [0083] (4) the amino acid sequence of position 85 to
position 169 in the amino acid sequence of any one of SEQ ID NOs:
1, 3, and 5; and [0084] (5) the amino acid sequence of position 89
to position 185 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5; [0085] [14] the composition of any one of
embodiments 1-13, wherein the peptide is a synthetic peptide;
[0086] [15] the composition of any one of embodiments 1-14, wherein
the peptide is a peptide produced using a cell; [0087] [16] the
composition of any one of embodiments 1-15, wherein the peptide is
a peptide to which a tag is added; [0088] [17] the composition of
any one of embodiments 1-15, wherein the peptide is a peptide to
which a tag-derived peptide fragment is added; [0089] [18] a
peptide that consists of a portion of an HMGB1 protein and has an
activity of stimulating migration of a cell; [0090] [19] the
peptide of embodiment 18, which is a peptide consisting of the
whole or part of the amino acid sequence of positions 1 to 195 or
positions 1 to 185 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5, and having an activity of stimulating migration
of a cell; [0091] [20] the peptide of embodiment 18, which
comprises any of the amino acid sequences below and has an activity
of stimulating migration of a cell: [0092] (1) the amino acid
sequence of position 17 to position 25 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5; [0093] (2) the amino acid
sequence of position 45 to position 74 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5; [0094] (3) the amino acid
sequence of position 55 to position 84 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5; [0095] (4) the amino acid
sequence of position 85 to position 169 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5; [0096] (5) the amino acid
sequence of position 89 to position 185 in the amino acid sequence
of any one of SEQ ID NOs: 1, 3, and 5; [0097] [21] the peptide of
embodiment 18, which is a peptide having an activity of stimulating
migration of a cell which consists of the whole or part of the
amino acid sequence of positions 1 to 195 or positions 1 to 185 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5, and
comprises any of the amino acid sequences below: [0098] (1) the
amino acid sequence of position 17 to position 25 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0099] (2) the
amino acid sequence of position 45 to position 74 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0100] (3) the
amino acid sequence of position 55 to position 84 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0101] (4) the
amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; [0102] (5) the
amino acid sequence of position 89 to position 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; [0103] [22] a
peptide that comprises any of the amino acid sequences below and
has an activity of stimulating migration of a cell: [0104] (1) the
amino acid sequence of position 17 to position 25 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0105] (2) the
amino acid sequence of position 45 to position 74 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0106] (3) the
amino acid sequence of position 55 to position 84 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0107] (4) the
amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; [0108] (5) the
amino acid sequence of position 89 to position 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; [0109] [23]
the peptide of any one of embodiments 18-22, which is a synthetic
peptide; [0110] [24] the peptide of any one of embodiments 18-22,
which is a peptide produced using a cell; [0111] [25] the peptide
of any one of embodiments 18-22, which is a peptide to which a tag
is added; [0112] [26] the peptide of any one of embodiments 18-22,
which is a peptide to which a tag-derived peptide fragment is
added; [0113] [27] a DNA encoding the peptide of any one of
embodiments 18-26; [0114] [28] a vector comprising the DNA of
embodiment 27; and [0115] [29] a transformed cell comprising the
DNA of embodiment 27 or the vector of embodiment 28.
BRIEF DESCRIPTION OF THE DRAWINGS
[0116] FIG. 1 shows an expression vector for producing peptides and
proteins using HEK293 cells.
[0117] FIG. 2A is a photograph showing the migration activity of an
established PDGFR.alpha.-positive bone marrow mesenchymal stem cell
line towards peptides. Comparisons were made over the positive
control full-length HMGB1 (1-215), a peptide consisting of amino
acids of positions 1 to 84 (1-84), a peptide consisting of amino
acids of positions 85 to 169 (85-169), a peptide consisting of
amino acids of positions 1 to 44 (1-44), a peptide consisting of
amino acids of positions 45 to 84 (45-84). All of these peptides
were produced using HEK293.
[0118] FIG. 2B shows Western blot confirming whether PDGFR.alpha.
was expressed in bone marrow mesenchymal stem cells or not.
PDGFR.alpha. expression in human bone marrow mesenchymal stem cells
was confirmed.
[0119] FIG. 3 is a photograph showing migration activity of an
established PDGFR.alpha.-positive bone marrow mesenchymal stem cell
line towards peptides. A comparison was made between a peptide
consisting of amino acids of positions 45 to 215 (45-215) of HMGB1
and a peptide consisting of amino acids of positions 63 to 215
(63-215). These peptides were all produced using HEK293.
[0120] FIG. 4 is a set of photographs showing GFP fluorescence of
CD11b-positive and CD11b-negative cells isolated by using MACS
after harvesting bone marrow cells from a PDGFR.alpha.-GFP mouse,
and culturing them for a certain period of time using adherent cell
culture dishes.
[0121] FIG. 5 is a photograph showing migration activity of primary
cultured bone marrow mesenchymal cells towards the HMGB1_1-44
peptide.
[0122] FIGS. 6A-6B are a set of photographs showing the bone
differentiation ability (FIG. 6A) and adipocyte differentiation
ability (FIG. 6B) of primary culture bone marrow mesenchymal cells
(PDGFR.alpha.-positive, Lin-negative, and c-kit negative).
[0123] FIG. 7 is a set of photographs showing migration activity of
an established PDGFR.alpha.-positive bone marrow mesenchymal stem
cell line towards various synthetic peptides.
[0124] FIG. 8A is a set of photographs showing migration activity
of an established PDGFR.alpha.-positive bone marrow mesenchymal
stem cell line towards various synthetic peptides.
[0125] FIG. 8B is a graph showing the quantified migration activity
in the left lower photograph in FIG. 8A. The number of cells that
migrated towards each of the synthetic peptides and the negative
control was measured under the microscope. The values were each
graphed with the average value of the negative control set to
100.
[0126] FIG. 8C is a photograph showing migration-promoting activity
of each peptide on bone marrow mesenchymal stem cells (MSC-1). The
graph shows the ratio of average number of cells measured for each
spot in the photograph relative to the negative control.
[0127] FIG. 9 is a photograph showing migration activity of an
established PDGFR.alpha.-positive bone marrow mesenchymal stem cell
line towards various synthetic peptides.
[0128] FIG. 10 is a photograph and diagram showing migration
activity of an established PDGFR.alpha.-positive bone marrow
mesenchymal stem cell line towards various peptides. The HMGB1_1-44
peptide (1-44) produced by HEK293 cells with constant peptide
secretion, HEK293 cells with peptide secretion through transient
plasmid transfection, E. coli, and peptide synthesis, were compared
with the positive control full-length HMGB1.
[0129] FIG. 11 shows FACS analysis of PDGFR.alpha., lineage marker,
and CD44 in an established PDGFR.alpha.-positive bone marrow
mesenchymal stem cells.
[0130] FIG. 12A is a photograph showing migration activity of mouse
keratinocytes towards the HMGB1_1-34 peptide.
[0131] FIG. 12B is a set of photographs of immunohistochemistry of
keratin 5 in PDGFR.alpha.-GFP mouse skin. Keratinocytes which are
keratin 5-positive cells did not express PDGFR.alpha..
[0132] FIG. 13A is a photograph showing migration activity of mouse
dermal fibroblasts towards the HMGB1_1-34 peptide (1-34).
[0133] FIG. 13B is a set of immunohistochemistry photographs of
vimentin in PDGFR.alpha.-GFP mouse skin. Some of the dermal
fibroblasts, which are vimentin-positive, expressed
PDGFR.alpha..
[0134] FIG. 14 shows FACS analysis of the PDGFR.alpha.-GFP mouse
dermal fibroblasts and the wild-type mouse (C57/B16 mouse) dermal
fibroblasts. Nearly 98% and more of the mouse dermal fibroblasts
expressed PDGFR.alpha..
[0135] FIG. 15 shows mobilization of PDGFR.alpha.-positive
CD44-positive cells into blood by a synthetic peptide (1-44)
demonstrated by FACS.
[0136] FIG. 16 is a cross-sectional photograph of the rat cerebral
infarction model administered with a synthetic peptide (1-44) or a
negative control, PBS. Reduction of the cerebral infarct size by
the synthetic peptide (1-44) was observed.
[0137] FIG. 17 is a set of diagrams and a photograph showing the
ratio of cerebral infarct lesion area relative to the right brain
area of the rat cerebral infarct model administered with a
synthetic peptide (1-44) or a negative control, PBS. Four cross
sections were produced from the same brain, and their respective
areas were measured.
[0138] FIG. 18A shows addition of a 6.times. His tag and a TEV
protease cleavage sequence to the N terminus of human HMGBE A cDNA
expressing this protein was newly made and inserted into an E. coli
expression vector.
[0139] FIG. 18B is a photograph showing migration activity of an
established PDGFR.alpha.-positive bone marrow mesenchymal stem cell
line towards HMGB1 fragments. The fragments were all produced using
E. coli.
[0140] FIG. 18C is a diagram obtained by quantifying migration
activity of an established PDGFR.alpha.-positive bone marrow
mesenchymal stem cell line towards the HMGB1 fragments, and
graphing the average values of the respective activities.
[0141] FIG. 18D is a table showing the average values of the
quantified migration activity of an established
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line
towards the HMGB1 fragments.
[0142] FIG. 19A is an SDS-PAGE photograph of fractions obtained by
anion exchange column purification of an HMGB1 fragment consisting
of amino acids of positions 89 to 215 that was produced using E.
coli and subjected to nickel affinity purification (I: input). M is
a molecular weight marker. A 15.5 kDa fragment (*3) was eluted
under a low salt concentration; and a 16 kDa fragment (*2) and a 17
kDa fragment (*1) were eluted in order as the salt concentration
increased. (*3) and (*2) are presumed to be degradation products of
(*1).
[0143] FIG. 19B is a photograph showing migration-promoting
activity of the fractions obtained in Fig. A on an established
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line. NC is
negative control, and 2-215 is positive control. The fragment with
the lowest molecular weight (*3), which was considered to be a
cleaved fragment, was found to have a stronger activity than longer
fragments (*1) and (*2). The activity was greater than that of
2-215.
[0144] FIG. 19C is an SDS-PAGE photograph of fractions obtained by
anion exchange column of an HMGB1 fragment consisting of amino
acids of positions 89 to 205 that was produced using E. coli and
subjected to nickel affinity purification (I: input). The shortest
fragment (*4) was eluted under a low salt concentration; and as the
salt concentration increased, longer fragments (*5) and (*6) were
eluted. (*5) and (*6) are predicted to be degradation products of
(*4). Also, purified HMGB1 fragments 89-195 and 89-185 were run on
SDS-PAGE at the same time. M is a molecular weight marker.
[0145] FIG. 19D is a photograph showing migration-promoting
activity of the fractions obtained in Fig. C on an established
PDGFR.alpha.-positive bone marrow mesenchymal stem cell line. NC is
negative control. The fragment with the lowest molecular weight
(*4), which was considered to be a cleaved fragment, was found to
have a stronger activity than longer fragments (*5) and (*6).
Meanwhile, HMGB1 fragments whose C terminus was further shortened
in advance, i.e. 89-195 and 89-185, showed a much stronger
activity.
[0146] FIG. 20A is a photograph showing migration-promoting
activity of the HMGB1 fragment 85-169. A stronger activity than
that of the positive control, HMGB1 fragment 2-215, was
observed.
[0147] FIG. 20B is a graph of average values of the quantified
migration activities in FIG. 20A.
[0148] FIG. 20C is a table showing the average values in FIG.
20B.
[0149] FIG. 21A shows migration activity of bone marrow mesenchymal
stem cell line (MSC-1) towards HMGB1 fragments produced using E.
coli: 2-215, 2-205, 2-195, and 2-185.
[0150] FIG. 21B is a photograph showing migration-promoting
activity of HMGB1 fragments produced using E. coli on human bone
marrow mesenchymal stem cells.
[0151] FIG. 21C shows a CBB protein staining of a gel on which
fusion fragments (2-84)+(186-215), (2-84)+(186-205), and
(2-84)+(186-195), which were obtained by adding to the purified
human HMGB1 fragment (2-84) a fragment of the acidic tail of human
HMGB1 ((186-215), (186-205), or (186-195)), were electrophoresed by
SDS-PAGE. Each purified fragment was confirmed.
[0152] FIG. 21D is a diagram in which migration-promoting activity
on MSC-1 was examined using the purified fragments.
Migration-promoting activity was not shown for any of the fusion
fragments obtained by adding an acidic tail sequence to the 2-84
fragment. Meanwhile, the 2-84 fragment itself showed
migration-promoting activity.
[0153] FIG. 22 is a set of photographs showing the skin flap made
on the back of a rat, which were taken one week later. PBS is the
negative control group. Groups administered with HMGB1 containing
the full length produced by HEK293 cells (1-215 (HEK)) and
administered with a synthetic peptide of amino acids of positions 1
to 44 (1-44 (synthetic peptide)) were compared. The arrows show
necrosed skin tissue.
[0154] FIG. 23 is a set of photographs showing the skin flap made
on the back of a rat, which were taken five weeks later. PBS is the
negative control group. Groups administered with HMGB1 containing
the full length produced in HEK293 cells (1-215 (HEK)) and
administered with a synthetic peptide of amino acids of positions 1
to 44 (1-44 (synthetic peptide)) were compared. The red-colored
sections are where skin ulcers were formed.
[0155] FIG. 24 is a set of photographs showing the skin flap made
on the back of a rat, which were taken seven weeks later. PBS is
the negative control group. Groups administered with HMGB1
containing the full length produced in HEK293 cells (1-215 (HEK))
and administered with a synthetic peptide of amino acids of
positions 1 to 44 (1-44 (synthetic peptide)) were compared.
[0156] FIG. 25 is a graph showing the quantified area of wound
section (necrosed section) that developed in the skin flap made on
the rat back. An effect of shrinking the wound section was
confirmed in the groups administered with HMGB1 containing the full
length produced in HEK293 cells (1-215 (HEK)) and administered with
a synthetic peptide of amino acids of positions 1 to 44 (1-44
(synthetic peptide)) in comparison with the negative control group
one to three weeks after the skin flap was made. After four weeks
and later, a further shrinking effect was observed in 1-44
(synthetic peptide) in comparison with the other two groups.
[0157] FIG. 26 is chemically synthesized HMGB1 peptides (1-44) and
(17-25) were administered to the caudal vein of a rat with produced
skin lesion. The figure shows the respective percentages of the
area of the skin lesion site relative to the entire area of the
skin flap two weeks and six weeks after the skin flap was made.
MODES FOR CARRYING OUT THE INVENTION
[0158] The present invention provides compositions for use in
stimulating cell migration, comprising a substance of any one of
(a) to (c) below: [0159] (a) a peptide consisting of a portion of
an HMGB1 protein and having cell migration-stimulating activity;
[0160] (b) a cell secreting the peptide of (a); and [0161] (c) a
vector into which a DNA encoding the peptide of (a) is
inserted.
[0162] The compositions used for stimulating cell migration in the
present invention include reagent compositions and pharmaceutical
compositions. In the present specification, reagent compositions
are also expressed as reagents, and pharmaceutical compositions are
also expressed as pharmaceuticals, agents, or pharmaceutical
compositions.
[0163] Reagent compositions used for stimulating cell migration in
the present invention can be used as reagents needed for basic
research and clinical research in, for example, regenerative
medicine and development of regeneration-inducing medicine. For
example, such reagent compositions can be used to recruit cells to
a living tissue in experimental animals, and then evaluate the
levels of tissue repair and tissue function reconstruction.
Further, such reagent compositions can be used to carry out in
vitro research on tissue regeneration by cell recruitment.
[0164] Pharmaceutical compositions used for stimulating cell
migration in the present invention can be used as pharmaceuticals
in, for example, regenerative medicine and regeneration-inducing
medicine. For example, such pharmaceutical compositions can be used
for tissue regeneration. Also, for example, such pharmaceutical
compositions can be used as so-called preventive drugs to prevent
the impairment of tissue and organ function due to reduction of
tissue stem cells, or alternatively as anti-aging drugs to delay
the progression of age-related changes.
[0165] In the present specification, compositions used for
stimulating cell migration are also expressed as agents used for
stimulating cell migration, cell migration-stimulating agents,
compositions used for inducing cell migration, agents used for
inducing cell migration, cell-migration-inducing agents, or
cell-attracting agents.
[0166] In the present invention, cell-migration-stimulating
activity refers to an activity to stimulate cell migration. In the
present specification, cell-migration-stimulating activity is also
expressed as cell-migration-inducing activity or cell-attracting
activity.
[0167] The present invention provides compositions for use in
mobilizing bone marrow cells from bone marrow to peripheral blood,
comprising a substance of any one of (a) to (c) below: [0168] (a) a
peptide consisting of a portion of an HMGB1 protein and having
cell-migration-stimulating activity; [0169] (b) a cell secreting
the peptide of (a); and [0170] (c) a vector into which a DNA
encoding the peptide of (a) is inserted.
[0171] The compositions used for mobilizing bone marrow cells from
bone marrow to peripheral blood in the present invention include
reagent compositions and pharmaceutical compositions.
[0172] Reagent compositions used for tissue regeneration in the
present invention can be used as reagents needed for basic research
and clinical research in, for example, regenerative medicine and
development of regeneration-inducing medicine. Pharmaceutical
compositions used for tissue regeneration in the present invention
can be used as pharmaceuticals in, for example, regenerative
medicine and regenerative-inducing medicine. For example, such
pharmaceutical compositions can be used to recruit bone marrow
tissue stem cells into peripheral circulation and regenerate
tissues. Further, it is also possible to collect cells ex vivo that
have been recruited into the peripheral blood using said
pharmaceutical compositions, and then administer the concentrated
cells to a tissue for treatment. Conventional methods are invasive
to the living body because cells are collected from the bone marrow
which is in the deep part of the body; however, when the
pharmaceutical compositions of the present invention are used, bone
marrow cells can be collected from peripheral blood less
invasively, and used in bone marrow cell transplantation. In the
present specification, compositions used for mobilizing bone marrow
cells from bone marrow to peripheral blood can be expressed as
compositions used for attracting bone marrow cells from bone marrow
to peripheral blood.
[0173] The present invention provides compositions used for tissue
regeneration, comprising a substance of any one of (a) to (c)
below: [0174] (a) a peptide consisting of a portion of an HMGB1
protein and having cell-migration-stimulating activity; [0175] (b)
a cell secreting the peptide of (a); and [0176] (c) a vector into
which a DNA encoding the peptide of (a) is inserted.
[0177] The compositions used for tissue regeneration in the present
invention include reagent compositions and pharmaceutical
compositions.
[0178] Reagent compositions used for tissue regeneration in the
present invention can be used as reagents needed for basic research
and clinical research in, for example, regenerative medicine and
development of regeneration-inducing medicine. Pharmaceutical
compositions used for tissue regeneration in the present invention
can be used as pharmaceuticals in, for example, regenerative
medicine and regeneration-inducing medicine.
[0179] In the present specification, compositions used for tissue
regeneration are also expressed as compositions used for inducing
or promoting tissue regeneration, agents used for inducing or
promoting tissue regeneration, tissue regeneration-inducing agents
or tissue regeneration-promoting agents. Tissue regeneration also
includes tissue repair.
[0180] Compositions used for tissue regeneration in the present
invention can be administered/added to any sites. That is, the
compositions can exert their effects no matter which tissue they
are administered to, such as a tissue in need of regeneration, a
tissue other than a tissue in need of regeneration, or blood. For
example, when the compositions are administered/added, cells are
recruited to the site of administration/addition or its nearby
tissue, thereby inducing or promoting tissue regeneration. Also,
for example, when the compositions are administered/added to a
damaged tissue site or its nearby region, cells are recruited to
the damaged tissue, thereby inducing or promoting tissue
regeneration. Further, for example, when the compositions are
administered/added to a tissue other than a tissue in need of
regeneration, bone marrow cells are mobilized from bone marrow to
the tissue in need of regeneration through peripheral circulation,
thereby inducing or promoting tissue regeneration. Here,
"peripheral circulation" is also called "blood circulation" or
"peripheral circulation bloodstream".
[0181] The tissue in need of regeneration includes, for example,
damaged tissues, necrotic tissues, tissues after surgery, tissues
with reduced function, fibrosing tissues, aged tissues, and
diseased tissues. Examples of the tissues include live skin tissues
and tissues obtained by internal biopsy (surgery) (brain, lung,
heart, liver, stomach, small intestine, large intestine, pancreas,
kidney, urinary bladder, spleen, uterus, testis, blood, etc.).
[0182] Administration to a tissue other than a tissue in need of
regeneration refers to administration to a site that is not a site
in need of regeneration (a site other than a site in need of
regeneration). Accordingly, "a tissue other than a tissue in need
of regeneration" can also be referred to as:
[0183] a site other than a tissue in need of regeneration; a site
other than a site in need of regeneration; a site distant from a
tissue in need of regeneration; a site distant from a site in need
of regeneration; a site distal to a site in need of regeneration; a
tissue distal to a tissue in need of regeneration; a distal site;
or a distal tissue.
[0184] Thus, compositions of the present invention are effectively
used to regenerate tissues (brain, heart, etc.) to which it is
difficult to directly administer pharmaceutical agents from outside
of the body.
[0185] Cells recruited to a tissue in need of regeneration
differentiate into various types of cells to contribute to
functional regeneration of the tissue in need of regeneration and
maintenance/enhancement of the functions. In the present invention,
examples of tissue in need of regeneration include, but are not
limited to, tissues damaged by various pathological conditions due
to ischemic/hypoperfusive/hypoxic conditions, trauma, burns,
inflammation, autoimmunity, gene abnormalities, and the like.
[0186] Tissues in the present invention are not particularly
limited as long as they are tissues into which bone marrow-derived
cells can differentiate. Examples include all types of tissues in
the living body, such as skin tissue, bone tissue, cartilage
tissue, muscle tissue, adipose tissue, cardiac muscle tissue,
neurological tissue, pulmonary tissue, gastrointestinal tissues,
hepatic/biliary/pancreatic tissues, and genitourinary organs.
Moreover, with use of the above compositions, treatments for
inducing functional tissue regeneration becomes possible not only
in cutaneous diseases such as intractable cutaneous ulcers, skin
wounds, bullosis, and alopecia, but also in tissues in need of
regeneration such as cerebral infarction, myocardial infarction,
bone fracture, pulmonary infarction, gastric ulcers, and enteritis.
Animal species to be administered with the above compositions are
not particularly limited, and include mammals, birds, fish, and
such. Mammals include human and non-human animals, which can be
exemplified by, but are not limited to, humans, mice, rats,
monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep,
and whales.
[0187] Examples of the tissue other than a tissue in need of
regeneration include blood tissues, muscle tissues, subcutaneous
tissues, intradermal tissues, abdominal cavity, and such.
[0188] Nerve tissues include central nervous tissues, but are not
limited thereto. Compositions used for regenerating nerve tissues
can be used to treat, for example, without limitation, cerebral
infarction, brain hemorrhage, and brain contusion. Compositions
used for regenerating bone tissues can be used to treat, for
example, without limitation, bone fracture. In addition,
compositions used for regenerating skin tissues can be used to
treat, for example, without limitation, skin ulcers, insufficient
suture closure of surgical wounds, burns, cuts, bruises, skin
erosions, and abrasions.
[0189] In the present invention, cells that are stimulated to
migrate or cells mobilized from bone marrow to peripheral blood
include undifferentiated cells and cells in various stages of
differentiation, but are not limited thereto. In the present
invention, cells that are stimulated to migrate or cells mobilized
from bone marrow to peripheral blood include stem cells, non-stem
cells, and such, but are not limited thereto. Stem cells include
circulatory stem cells and non-circulatory stem cells.
Non-circulatory stem cells are, for example, stem cells residing in
a tissue. Circulatory stem cells are, for example, circulatory stem
cells in blood.
[0190] Further, cells stimulated to migrate or cells mobilized from
bone marrow to peripheral blood include bone marrow-derived cells
and hematopoietic stem cells, but are not limited thereto. In the
present specification, "hematopoietic stem cells" are stem cells
that can differentiate into blood cells such as red blood cells,
platelets, mast cells, and dendritic cells, as well as white blood
cells including neutrophils, eosinophils, basophils, lymphocytes,
monocytes, macrophages, and such. Their markers are known to be
CD34-positive and CD133-positive in human, and CD34-negative,
c-Kit-positive, Sca-1-positive, and lineage marker-negative in
mouse. Hematopoietic stem cells are difficult to be cultured alone
when cultured in culture dishes, and they need to be co-cultured
with stromal cells.
[0191] In the present specification, "bone marrow cells" means
cells present inside bone marrow while "bone marrow-derived cells"
means "bone marrow cells" mobilized from bone marrow to outside of
bone marrow. "Bone marrow cells" include cells containing tissue
progenitor cell populations present inside bone marrow. Further,
"bone marrow-derived cells" may be cells containing mesoangioblasts
or cells free of mesoangioblasts.
[0192] Tissue progenitor cells are defined as undifferentiated
cells having a unidirectional potency to differentiate into cells
of a specific tissue other than the blood system, and include
undifferentiated cells having the potency to differentiate into
mesenchymal tissues, epithelial tissues, nerve tissues,
parenchymatous organs, and vascular endothelium as mentioned
above.
[0193] "Bone marrow cells" and "bone marrow-derived cells" are
hematopoietic stem cells and differentiated cells derived therefrom
such as leukocytes, erythrocytes, platelets, osteoblasts, and
fibrocytes, or are stern cells represented by cells which have been
hitherto called bone marrow mesenchymal stem cells, bone marrow
stromal pluripotent stem cells, or bone marrow pluripotent stem
cells. As used herein, "bone marrow stem cells" refer to stem cells
present inside bone marrow, while "bone marrow-derived stem cells"
refer to "bone marrow stem cells" mobilized from bone marrow to
outside of bone marrow. In the present invention, cells stimulated
to migrate or mobilized from bone marrow to peripheral blood
include "bone marrow-derived stem cells", but are not limited
thereto. "Bone marrow cells" and "bone marrow-derived cells" can be
isolated by bone marrow collection (bone marrow cell collection) or
peripheral blood collection. Hematopoietic stem cells are
nonadherent, while some of the "bone marrow cells" and "bone
marrow-derived cells" are obtained as adherent cells by means of a
cell culture of a monocyte fraction of blood obtained by the bone
marrow collection (bone marrow cell collection) or peripheral blood
collection.
[0194] Moreover, "bone marrow cells" and "bone marrow-derived
cells" include mesenchymal stem cells, and have a potential to
differentiate into, preferably, osteoblasts (which can be
identified by observing calcification after inducing
differentiation), chondrocytes (which can be identified by alcian
blue positive staining, safranin O positive staining, or the like),
adipocytes (which can be identified by Sudan III positive
staining), and other mesenchymal cells such as fibroblasts, smooth
muscle cells, stromal cells, and tendon cells; and further nerve
cells, epithelial cells (for example, epidermal keratinocytes and
intestinal epithelial cells express cytokeratin family), and
vascular endothelial cells. The cells to be differentiated into are
not limited to the above cells, and the potential to differentiate
into cells of parenchymatous organs such as liver, kidney, and
pancreas is also included.
[0195] Herein, "bone marrow mesenchymal stem cells", "bone marrow
stromal pluripotent cells" or "bone marrow pluripotent stem cells"
refer to cells existing in the bone marrow, which are directly
collected from the bone marrow or indirectly collected from other
tissues (blood, skin, fat, and other tissues), and can be cultured
and proliferated as adherent cells on a culture dish (made of
plastic or glass). These cells are characterized in having a
potential to differentiate into mesenchymal tissues such as bone,
cartilage, and fat (mesenchymal stem cells), or into skeletal
muscle, heart muscle, nervous tissues, and epithelial tissues
(pluripotent stem cells), and can be obtained by collection of bone
marrow cells.
[0196] On the other hand, "bone marrow-derived bone marrow
mesenchymal stem cells", "bone marrow-derived bone marrow stromal
pluripotent cells", or "bone marrow-derived bone marrow pluripotent
stem cells" mobilized from bone marrow to outside of the bone
marrow are cells that can be obtained by collection from peripheral
blood, mesenchymal tissues such as fat, epithelial tissues such as
skin, or nervous tissues such as brain.
[0197] In addition, these cells are also characterized in having a
potential to differentiate into epithelial tissues such as
keratinocytes that constitute skin, or nervous tissues that
constitute brain, when administered to a lesion area of the living
body immediately after collection or after once being adhered onto
a culture dish.
[0198] Bone marrow mesenchymal stem cells, bone marrow stromal
pluripotent stem cells, bone marrow pluripotent stem cells, or
these cells recruited from bone marrow to outside of the bone
marrow preferably have a potency to differentiate into: osteoblasts
(which can be identified by observing calcification after inducing
differentiation), chondrocytes (which can be identified by alcian
blue positive staining, safranin O positive staining, or the like),
adipocytes (which can be identified by Sudan III positive
staining), and other mesenchymal cells such as fibroblasts, smooth
muscle cells, skeletal muscle cells, stromal cells, and tendon
cells; nerve cells, pigment cells, epidermal cells, hair follicle
cells (which express cytokeratin family, hair keratin family, or
the like), epithelial cells (for example, epidermal keratinocytes
and intestinal epithelial cells express cytokeratin family or the
like), and endothelial cells; and further preferably into cells of
parenchymatous organs such as liver, kidney, and pancreas. However,
differentiated cells are not limited to the above cells.
[0199] Human bone marrow cells and human bone marrow-derived cells
can be exemplified by, but are not limited to, cells which can be
directly obtained by collecting bone marrow (cells), peripheral
blood, or fat, or obtained as adherent cells through culturing of
an isolated monocyte fraction. Markers for human bone marrow cells
and human bone marrow-derived cells include, for example, all or
some of the following but are not limited thereto:
PDGFR.alpha.-positive, Lin-negative, CD45-negative, CD44-positive,
CD90-positive, and CD29-positive, Flk-1-negative, CDI05-positive,
CD73-positive, CD90-positive, CD71-positive, Stro-1-positive,
CD106-positive, CD166-positive, and CD31-negative.
[0200] Moreover, mouse bone marrow cells and mouse bone
marrow-derived cells can be exemplified by, but are not limited to,
cells which can be directly obtained by collecting bone marrow
(cells), peripheral blood, or fat, or obtained as adherent cells
through culturing of an isolated monocyte fraction. Markers for
mouse bone marrow cells and mouse bone marrow-derived cells
include, for example, all or some of the following but are not
limited thereto: CD44-positive, PDGFR.alpha.-positive,
PDGFR.beta.-positive, CD45-negative, Lin-negative, Sea-1 positive,
c-kit negative, CD90-positive, CD29-positive, and
Flk-1-negative.
[0201] In the present invention, cells stimulated to migrate or
mobilized from bone marrow to peripheral blood are, for example,
PDGFR.alpha.-positive cells, but are not limited thereto. Further,
markers other than PDGFR.alpha. can be exemplified by all or some
of CD29-positive, CD44-positive, CD90-positive, CD271-positive,
CD11b-negative, and Flk-1-negative, but are not limited thereto.
PDGFR.alpha.-positive cells include, but are not limited to, for
example, PDGFR.alpha.-positive bone marrow-derived cells,
PDGFR.alpha.-positive bone marrow-derived bone marrow mesenchymal
stem cells, tissue cells residing in PDGFR.alpha.-positive tissues
(for example, fibroblasts and such), PDGFR.alpha.-positive bone
marrow-derived cells obtained as adherent cells by means of cell
culture of a monocyte fraction of blood obtained by bone marrow
collection (bone marrow cell collection) or peripheral blood
collection.
[0202] Compositions of the present invention may contain substances
other than at least one of the substances (a) to (c) mentioned
above. In the compositions of the present invention, there is no
particular limitation in substances other than at least one of the
substances (a) to (c) mentioned above, so long as they do not
inhibit the cell migration-stimulating activity, cell mobilization
activity, or tissue regeneration promoting activity. For example,
in addition to at least one of the substances (a) to (c) mentioned
above, the compositions of the present invention may contain:
related molecule(s) enhancing the function of substances (a) to (c)
mentioned above; molecule(s) which inhibit unanticipated actions of
substances (a) to (c) mentioned above; factors which regulate
proliferation and differentiation of cells;
[0203] and other factors which enhance/maintain these factors or
cellular functions.
[0204] The HMGB1 protein in the present invention includes, but is
not limited to, for example, a protein comprising the amino acid
sequence of SEQ ID NO: 1 as a human-derived HMGB1 protein, and DNA
encoding said protein includes, but is not limited to, for example,
a DNA comprising the nucleotide sequence of SEQ ID NO: 2.
[0205] Further, the mouse-derived HMGB 1 protein includes, but is
not limited to, for example, a protein comprising the amino acid
sequence of SEQ ID NO: 3, and DNA encoding said protein includes,
but is not limited to, for example, a DNA comprising the nucleotide
sequence of SEQ ID NO: 4.
[0206] Further, the rat-derived HMGB1 protein includes, but is not
limited to, for example, a protein comprising the amino acid
sequence of SEQ ID NO: 5, and DNA encoding said protein includes,
but not limited to, for example, a DNA comprising the nucleotide
sequence of SEQ ID NO: 6.
[0207] Compositions of the present invention comprise a peptide
consisting of a portion of an HMGB1 protein and having cell
migration-stimulating activity. The peptide consisting of a portion
of an HMGB1 protein of the present invention is not particularly
limited as long as it contains a domain having cell
migration-stimulating activity.
[0208] The cell migration-stimulating activity of a peptide
consisting of a portion of an HMGB1 protein can be verified by, for
example, methods described in the Examples and methods shown below,
without limitation; and it can also be measured using another in
vitro or in vivo method for measuring cell migration ability.
[0209] Method in which a silicone tube inserted with an HMGB1
protein or peptide is implanted under the skin and such, and taken
out after a certain period of time to observe cells that migrate
into the tube. [0210] Method in which resin beads and the like
bound to an HMGB1 protein or peptide are implanted in a body
tissue, and taken out after a certain period of time to observe
cells that migrate into the beads. [0211] Method in which polymers
that have a sustained release action, such as gelatin and
hyaluronic acid, are impregnated with an HMGB1 protein or peptide
and implanted in a body tissue, and taken out after a certain
period of time to observe cells that migrate into the polymers.
[0212] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity can be exemplified by the peptides below, but are not
limited thereto.
[0213] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes, for example, a peptide having an activity of
mobilizing cells from bone marrow to peripheral blood, or an
activity of promoting tissue regeneration.
[0214] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes, for example, a peptide consisting of the whole
or part of the amino acid sequence of positions 1 to 195 or
positions 1 to 185 in the amino acid sequence of any of SEQ ID NOs:
1, 3, and 5, and having cell migration-stimulating activity.
[0215] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes, for example, a peptide that comprises at least
any one of the amino acid sequences below and has cell
migration-stimulating activity. The following amino acid sequences
are part of the amino acid sequence of any of SEQ ID NOs: 1, 3, and
5: [0216] (1) the amino acid sequence of position 17 to position
25; [0217] (2) the amino acid sequence of position 45 to position
74; [0218] (3) the amino acid sequence of position 55 to position
84; [0219] (4) the amino acid sequence of position 85 to position
169; [0220] (5) the amino acid sequence of position 89 to position
185; and [0221] (6) the amino acid sequence of position 93 to
position 215.
[0222] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes a peptide having cell migration-stimulating
activity which consists of the whole or part of the amino acid
sequence of positions 1 to 195 or positions 1 to 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least any one of the amino acid sequences
below. The following amino acid sequences are part of the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5: [0223] (1) a
peptide comprising the amino acid sequence of position 17 to
position 25; [0224] (2) a peptide comprising the amino acid
sequence of position 45 to position 74; [0225] (3) a peptide
comprising the amino acid sequence of position 55 to position 84;
[0226] (4) a peptide comprising the amino acid sequence of position
85 to position 169; and [0227] (5) a peptide comprising the amino
acid sequence of position 89 to position 185.
[0228] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes a peptide having cell migration-stimulating
activity which consists of a portion of the amino acid sequence of
any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises
at least the amino acid sequence of position 17 to position 25 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 1 to 195 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 195), a peptide consisting of the whole or part of the
amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 185), a peptide consisting of the
whole or part of the amino acid sequence of positions 1 to 170 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5 (the
number of amino acids in said peptide is an amino acid number
selected from natural numbers less than or equal to 170), a peptide
consisting of the whole or part of the amino acid sequence of
positions 1 to 84 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5 (the number of amino acids in said peptide is an
amino acid number selected from natural numbers less than or equal
to 84), a peptide consisting of the whole or part of the amino acid
sequence of positions 1 to 44 in the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5 (the number of amino acids in said
peptide is an amino acid number selected from natural numbers less
than or equal to 44), wherein the peptide comprises at least the
amino acid sequence of positions 17 to 25 in said amino acid
sequence and has cell migration-stimulating activity.
[0229] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 1 to 195 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 17
to position 25 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, and wherein the
peptide comprises at least the amino acid sequence of position 17
to position 25 in said amino acid sequence", can also be described
similarly.
[0230] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 17 to
position 25 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 17 to
position 25 in said amino acid sequence".
[0231] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 17 to
position 25 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any one of (1) to (60) shown
below, and comprises at least the amino acid sequence of position
17 to position 25 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5.
[0232] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 17 to
position 25 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 1 to position 195 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (1) to (57) and (59) to (61) shown below in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0233] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 17 to
position 25 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (1) to (61) shown below. The following
amino acid sequences are part of the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5: [0234] (1) the amino acid sequence of
position 1 to position 44; [0235] (2) the amino acid sequence of
position 1 to position 25; [0236] (3) the amino acid sequence of
position 1 to position 34; [0237] (4) the amino acid sequence of
position 1 to position 42; [0238] (5) the amino acid sequence of
position 1 to position 43; [0239] (6) the amino acid sequence of
position 1 to position 45; [0240] (7) the amino acid sequence of
position 1 to position 46; [0241] (8) the amino acid sequence of
position 1 to position 47; [0242] (9) the amino acid sequence of
position 1 to position 48; [0243] (10) the amino acid sequence of
position 1 to position 49; [0244] (11) the amino acid sequence of
position 1 to position 50; [0245] (12) the amino acid sequence of
position 1 to position 51; [0246] (13) the amino acid sequence of
position 1 to position 52; [0247] (14) the amino acid sequence of
position 1 to position 62; [0248] (15) the amino acid sequence of
position 1 to position 84; [0249] (16) the amino acid sequence of
position 10 to position 25; [0250] (17) the amino acid sequence of
position 11 to position 25; [0251] (18) the amino acid sequence of
position 11 to position 27; [0252] (19) the amino acid sequence of
position 11 to position 28; [0253] (20) the amino acid sequence of
position 11 to position 29; [0254] (21) the amino acid sequence of
position 11 to position 30; [0255] (22) the amino acid sequence of
position 11 to position 34; [0256] (23) the amino acid sequence of
position 11 to position 44; [0257] (24) the amino acid sequence of
position 12 to position 25; [0258] (25) the amino acid sequence of
position 12 to position 30; [0259] (26) the amino acid sequence of
position 13 to position 25; [0260] (27) the amino acid sequence of
position 13 to position 30; [0261] (28) the amino acid sequence of
position 14 to position 25; [0262] (29) the amino acid sequence of
position 14 to position 30; [0263] (30) the amino acid sequence of
position 15 to position 25; [0264] (31) the amino acid sequence of
position 15 to position 30; [0265] (32) the amino acid sequence of
position 16 to position 25; [0266] (33) the amino acid sequence of
position 16 to position 30; [0267] (34) the amino acid sequence of
position 17 to position 30; [0268] (35) the amino acid sequence of
position 1 to position 70; [0269] (36) the amino acid sequence of
position 1 to position 81; [0270] (37) the amino acid sequence of
position 1 to position 170; [0271] (38) the amino acid sequence of
position 2 to position 25; [0272] (39) the amino acid sequence of
position 2 to position 34; [0273] (40) the amino acid sequence of
position 2 to position 42; [0274] (41) the amino acid sequence of
position 2 to position 43; [0275] (42) the amino acid sequence of
position 2 to position 44; [0276] (43) the amino acid sequence of
position 2 to position 45; [0277] (44) the amino acid sequence of
position 2 to position 46; [0278] (45) the amino acid sequence of
position 2 to position 47; [0279] (46) the amino acid sequence of
position 2 to position 48; [0280] (47) the amino acid sequence of
position 2 to position 49; [0281] (48) the amino acid sequence of
position 2 to position 50; [0282] (49) the amino acid sequence of
position 2 to position 51; [0283] (50) the amino acid sequence of
position 2 to position 52; [0284] (51) the amino acid sequence of
position 2 to position 62; [0285] (52) the amino acid sequence of
position 2 to position 70; [0286] (53) the amino acid sequence of
position 2 to position 81; [0287] (54) the amino acid sequence of
position 2 to position 84; [0288] (55) the amino acid sequence of
position 2 to position 170; [0289] (56) the amino acid sequence of
position 17 to position 44; [0290] (57) the amino acid sequence of
position 1 to position 185; [0291] (58) the amino acid sequence of
position 1 to position 195; [0292] (59) the amino acid sequence of
position 2 to position 185; [0293] (60) the amino acid sequence of
position 2 to position 195; and [0294] (61) the amino acid sequence
of position 17 to position 25.
[0295] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes a peptide having cell migration-stimulating
activity which consists of a portion of the amino acid sequence of
any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises
at least the amino acid sequence of position 45 to position 74 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 1 to 195 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 195), a peptide consisting of the whole or part of the
amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 185), a peptide consisting of the
whole or part of the amino acid sequence of positions 1 to 170 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5 (the
number of amino acids in said peptide is an amino acid number
selected from natural numbers less than or equal to 170), a peptide
consisting of the whole or part of the amino acid sequence of
positions 1 to 84 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5 (the number of amino acids in said peptide is an
amino acid number selected from natural numbers less than or equal
to 84), a peptide consisting of the whole or part of the amino acid
sequence of positions 45 to 84 in the amino acid sequence of any
one of SEQ ID NOs: 1, 3, and 5 (the number of amino acids in said
peptide is an amino acid number selected from natural numbers less
than or equal to 40), wherein the peptide comprises at least the
amino acid sequence of positions 45 to 74 in said amino acid
sequence and has cell migration-stimulating activity.
[0296] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 1 to 195 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 45
to position 74 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity consisting of the whole or part of
the amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence", can also be described
similarly.
[0297] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence".
[0298] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any one of (a) to (k) below, and
comprises at least the amino acid sequence of position 45 to
position 74 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5.
[0299] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 1 to position 195 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (a) to (h) and (j) to (1) in the amino acid sequence of
any one of SEQ ID NOs: 1, 3, and 5.
[0300] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 45 to
position 74 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (a) to (1). The following amino acid
sequences are part of the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5: [0301] (a) the amino acid sequence of position 1
to position 84; [0302] (b) the amino acid sequence of position 45
to position 84; [0303] (c) the amino acid sequence of position 1 to
position 81; [0304] (d) the amino acid sequence of position 1 to
position 170; [0305] (e) the amino acid sequence of position 2 to
position 81; [0306] (f) the amino acid sequence of position 2 to
position 84; [0307] (g) the amino acid sequence of position 2 to
position 170; [0308] (h) the amino acid sequence of position 1 to
position 185; [0309] (i) the amino acid sequence of position 1 to
position 195; [0310] (j) the amino acid sequence of position 2 to
position 185; [0311] (k) the amino acid sequence of position 2 to
position 195; and [0312] (l) the amino acid sequence of position 45
to position 74.
[0313] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity is a peptide having cell migration-stimulating activity
which consists of a portion of the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises at least
the amino acid sequence of position 55 to position 84 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0314] Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 1 to 195 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 195), a peptide consisting of the whole or part of the
amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 185), a peptide consisting of the
whole or part of the amino acid sequence of positions 1 to 170 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5 (the
number of amino acids in said peptide is an amino acid number
selected from natural numbers less than or equal to 170), a peptide
consisting of the whole or part of the amino acid sequence of
positions 1 to 84 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5 (the number of amino acids in said peptide is an
amino acid number selected from natural numbers less than or equal
to 84), a peptide consisting of the whole or part of the amino acid
sequence of positions 45 to 84 in the amino acid sequence of any
one of SEQ ID NOs: 1, 3, and 5 (the number of amino acids in said
peptide is an amino acid number selected from natural numbers less
than or equal to 40), wherein the peptide comprises at least the
amino acid sequence of positions 55 to 84 in said amino acid
sequence and has cell migration-stimulating activity.
[0315] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 1 to 195 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 55
to position 84 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence", can also be described
similarly.
[0316] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence".
[0317] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any of (A) to (J) below, and
comprises at least the amino acid sequence of position 55 to
position 84 in the amino acid sequence of any one of SEQ ID NOs: 1,
3, and 5.
[0318] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 1 to position 195 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (A) to (G) and (I) to (K) below in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0319] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 55 to
position 84 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (A) to (K) below. The following amino
acid sequences are part of the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5: [0320] (A) the amino acid sequence of
position 1 to position 84; [0321] (B) the amino acid sequence of
position 45 to position 84; [0322] (C) the amino acid sequence of
position 1 to position 170; [0323] (D) the amino acid sequence of
position 2 to position 84; [0324] (F) a peptide comprising the
amino acid sequence of position 2 to position 170; [0325] (G) the
amino acid sequence of position 1 to position 185; [0326] (H) the
amino acid sequence of position 1 to position 195; [0327] (I) the
amino acid sequence of position 2 to position 185; [0328] (J) the
amino acid sequence of position 2 to position 195; and [0329] (K)
the amino acid sequence of position 55 to position 84.
[0330] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity is a peptide having cell migration-stimulating activity
which consists of a portion of the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises at least
the amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0331] Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 1 to 195 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 195), a peptide consisting of the whole or part of the
amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 185), a peptide consisting of the
whole or part of the amino acid sequence of positions 1 to 170 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5 (the
number of amino acids in said peptide is an amino acid number
selected from natural numbers less than or equal to 170), a peptide
consisting of the whole or part of the amino acid sequence of
positions 89 to 185 in the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5 (the number of amino acids in said peptide is an
amino acid number selected from natural numbers less than or equal
to 101), wherein the peptide comprises at least the amino acid
sequence of positions 85 to 169 in said amino acid sequence and has
cell migration-stimulating activity.
[0332] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 1 to 195 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 85
to position 169 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence", can also be described
similarly.
[0333] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence".
[0334] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any one of (i) to (vi) below,
and comprises at least the amino acid sequence of position 85 to
position 169 in the amino acid sequence of any one of SEQ ID NOs:
1, 3, and 5.
[0335] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 1 to position 195 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (i) to (iii) and (v) to (vii) below in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0336] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 85 to
position 169 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (i) to (vii) below. The following amino
acid sequences are part of the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5: [0337] (i) the amino acid sequence of
position 1 to position 170; [0338] (ii) the amino acid sequence of
position 2 to position 170; [0339] (iii) the amino acid sequence of
position 1 to position 185; [0340] (iv) the amino acid sequence of
position 1 to position 195; [0341] (v) the amino acid sequence of
position 2 to position 185; [0342] (vi) the amino acid sequence of
position 2 to position 195; and [0343] (vii) position 85 to
position 169.
[0344] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity includes a peptide having cell migration-stimulating
activity which consists of a portion of the amino acid sequence of
any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises
at least the amino acid sequence of position 89 to position 185 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 1 to 195 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 195), and a peptide consisting of the whole or part of the
amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 185), wherein the peptide comprises
at least the amino acid sequence of positions 89 to 185 in said
amino acid sequence and has cell migration-stimulating
activity.
[0345] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 1 to 195 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 89
to position 185 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 185 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence", can also be described
similarly.
[0346] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence".
[0347] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any one of (I) to (VI) below,
wherein the peptide comprises at least the amino acid sequence of
position 89 to position 185 in the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5.
[0348] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 1 to position 195 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (I) and (III) to (V) in the amino acid sequence of any
one of SEQ ID NOs: 1, 3, and 5.
[0349] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 1 to 195 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 185 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (I) to (V) below. The following amino
acid sequences are part of the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5: [0350] (I) the amino acid sequence of
position 1 to position 185; [0351] (II) the amino acid sequence of
position 1 to position 195; [0352] (III) the amino acid sequence of
position 2 to position 185; [0353] (IV) the amino acid sequence of
position 2 to position 195; and [0354] (V) the amino acid sequence
of position 89 to position 185.
[0355] In the present invention, the peptide consisting of a
portion of an HMGB1 protein and having cell migration-stimulating
activity is a peptide having cell migration-stimulating activity
which consists of a portion of the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5, wherein the peptide comprises at least
the amino acid sequence of position 93 to position 215 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0356] Examples of such peptides include, but are not limited to, a
peptide consisting of the whole or part of the amino acid sequence
of positions 45 to 215 in the amino acid sequence of any one of SEQ
ID NOs: 1, 3, and 5 (the number of amino acids in said peptide is
an amino acid number selected from natural numbers less than or
equal to 171), a peptide consisting of the whole or part of the
amino acid sequence of positions 63 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5 (the number of amino
acids in said peptide is an amino acid number selected from natural
numbers less than or equal to 153), and a peptide consisting of the
whole or part of the amino acid sequence of positions 89 to 215 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5 (the
number of amino acids in said peptide is an amino acid number
selected from natural numbers less than or equal to 123), wherein
the peptide comprises at least the amino acid sequence of positions
93 to 215 in said amino acid sequence and has cell
migration-stimulating activity.
[0357] The following description is regarding "a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of positions 45 to 215 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the
peptide comprises at least the amino acid sequence of position 93
to position 215 in said amino acid sequence". However, other
peptides included in said peptide, such as "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 63 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 89 to
position 215 in said amino acid sequence" can also be described
similarly.
[0358] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 45 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 93 to
position 215 in said amino acid sequence" may also be expressed as
"a peptide having cell migration-stimulating activity which
consists of a consecutive amino acid sequence selected from the
amino acid sequence of positions 45 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 93 to
position 215 in said amino acid sequence".
[0359] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 45 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 93 to
position 215 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of any one of (W) to (Y) below,
wherein the peptide comprises at least the amino acid sequence of
position 93 to position 215 in the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5.
[0360] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 45 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 93 to
position 215 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the whole or
part of the amino acid sequence of position 45 to position 215 in
the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5,
wherein the peptide comprises at least the amino acid sequence of
any one of (X) to (Z) below in the amino acid sequence of any one
of SEQ ID NOs: 1, 3, and 5.
[0361] In the present invention, "a peptide having cell
migration-stimulating activity which consists of the whole or part
of the amino acid sequence of positions 45 to 215 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5, wherein the peptide
comprises at least the amino acid sequence of position 93 to
position 215 in said amino acid sequence" includes a peptide having
cell migration-stimulating activity which consists of the amino
acid sequence of any one of (W) to (Z) below. The following amino
acid sequences are part of the amino acid sequence of any one of
SEQ ID NOs: 1, 3, and 5: [0362] (W) a peptide comprising the amino
acid sequence of position 45 to position 215; [0363] (X) a peptide
comprising the amino acid sequence of position 63 to position 215;
[0364] (Y) a peptide comprising the amino acid sequence of position
89 to position 215; and [0365] (Z) a peptide comprising the amino
acid sequence of position 93 to position 215.
[0366] Thus, the peptide consisting of a portion of an HMGB1
protein and having cell migration-stimulating activity in the
present invention includes, but is not limited to, the following
peptides: [0367] <1> a peptide comprising the amino acid
sequence of position 1 to position 44; [0368] <2> a peptide
comprising the amino acid sequence of position 1 to position 25;
[0369] <3> a peptide comprising the amino acid sequence of
position 1 to position 34; [0370] <4> a peptide comprising
the amino acid sequence of position 1 to position 42;
[0371] 35 [0372] <5> a peptide comprising the amino acid
sequence of position 1 to position 43; [0373] <6> a peptide
comprising the amino acid sequence of position 1 to position 45;
[0374] <7> a peptide comprising the amino acid sequence of
position 1 to position 46; [0375] <8> a peptide comprising
the amino acid sequence of position 1 to position 47; [0376]
<9> a peptide comprising the amino acid sequence of position
1 to position 48; [0377] <10> a peptide comprising the amino
acid sequence of position 1 to position 49; [0378] <11> a
peptide comprising the amino acid sequence of position 1 to
position 50; [0379] <12> a peptide comprising the amino acid
sequence of position 1 to position 51; [0380] <13> a peptide
comprising the amino acid sequence of position 1 to position 52;
[0381] <14> a peptide comprising the amino acid sequence of
position 1 to position 62; [0382] <15> a peptide comprising
the amino acid sequence of position 1 to position 84; [0383]
<16> a peptide comprising the amino acid sequence of position
10 to position 25; [0384] <17> a peptide comprising the amino
acid sequence of position 11 to position 25; [0385] <18> a
peptide comprising the amino acid sequence of position 11 to
position 27; [0386] <19> a peptide comprising the amino acid
sequence of position 11 to position 28; [0387] <20> a peptide
comprising the amino acid sequence of position 11 to position 29;
[0388] <21> a peptide comprising the amino acid sequence of
position 11 to position 30; [0389] <22> a peptide comprising
the amino acid sequence of position 11 to position 34; [0390]
<23> a peptide comprising the amino acid sequence of position
11 to position 44; [0391] <24> a peptide comprising the amino
acid sequence of position 12 to position 25; [0392] <25> a
peptide comprising the amino acid sequence of position 12 to
position 30; [0393] <26> a peptide comprising the amino acid
sequence of position 13 to position 25; [0394] <27> a peptide
comprising the amino acid sequence of position 13 to position 30;
[0395] <28> a peptide comprising the amino acid sequence of
position 14 to position 25; [0396] <29> a peptide comprising
the amino acid sequence of position 14 to position 30; [0397]
<30> a peptide comprising the amino acid sequence of position
15 to position 25; [0398] <31> a peptide comprising the amino
acid sequence of position 15 to position 30; [0399] <32> a
peptide comprising the amino acid sequence of position 16 to
position 25; [0400] <33> a peptide comprising the amino acid
sequence of position 16 to position 30; [0401] <34> a peptide
comprising the amino acid sequence of position 17 to position 25;
[0402] <35> a peptide comprising the amino acid sequence of
position 17 to position 30; [0403] <36> a peptide comprising
the amino acid sequence of position 45 to position 74; [0404]
<37> a peptide comprising the amino acid sequence of position
45 to position 84; [0405] <38> a peptide comprising the amino
acid sequence of position 45 to position 215; [0406] <39> a
peptide comprising the amino acid sequence of position 55 to
position 84; [0407] <40> a peptide comprising the amino acid
sequence of position 63 to position 215; [0408] <41> a
peptide comprising the amino acid sequence of position 1 to
position 70; [0409] <42> a peptide comprising the amino acid
sequence of position 1 to position 81; [0410] <43> a peptide
comprising the amino acid sequence of position 1 to position 170;
[0411] <44> a peptide comprising the amino acid sequence of
position 2 to position 25; [0412] <45> a peptide comprising
the amino acid sequence of position 2 to position 34; [0413]
<46> a peptide comprising the amino acid sequence of position
2 to position 42; [0414] <47> a peptide comprising the amino
acid sequence of position 2 to position 43; [0415] <48> a
peptide comprising the amino acid sequence of position 2 to
position 44; [0416] <49> a peptide comprising the amino acid
sequence of position 2 to position 45; [0417] <50> a peptide
comprising the amino acid sequence of position 2 to position 46;
[0418] <51> a peptide comprising the amino acid sequence of
position 2 to position 47; [0419] <52> a peptide comprising
the amino acid sequence of position 2 to position 48; [0420]
<53> a peptide comprising the amino acid sequence of position
2 to position 49; [0421] <54> a peptide comprising the amino
acid sequence of position 2 to position 50; [0422] <55> a
peptide comprising the amino acid sequence of position 2 to
position 51; [0423] <56> a peptide comprising the amino acid
sequence of position 2 to position 52; [0424] <57> a peptide
comprising the amino acid sequence of position 2 to position 62;
[0425] <58> a peptide comprising the amino acid sequence of
position 2 to position 70; [0426] <59> a peptide comprising
the amino acid sequence of position 2 to position 81; [0427]
<60> a peptide comprising the amino acid sequence of position
2 to position 84; [0428] <61> a peptide comprising the amino
acid sequence of position 2 to position 170; [0429] <62> a
peptide comprising the amino acid sequence of position 85 to
position 169; [0430] <63> a peptide comprising the amino acid
sequence of position 89 to position 185; [0431] <64> a
peptide comprising the amino acid sequence of position 89 to
position 195; [0432] <65> a peptide comprising the amino acid
sequence of position 89 to position 205; [0433] <66> a
peptide comprising the amino acid sequence of position 89 to
position 215; [0434] <67> a peptide comprising the amino acid
sequence of position 93 to position 215; [0435] <68> a
peptide comprising the amino acid sequence of position 17 to
position 44; [0436] <69> a peptide comprising the amino acid
sequence of position 1 to position 185; [0437] <70> a peptide
comprising the amino acid sequence of position 1 to position 195;
[0438] <71> a peptide comprising the amino acid sequence of
position 1 to position 205; [0439] <72> a peptide comprising
the amino acid sequence of position 2 to position 185; [0440]
<73> a peptide comprising the amino acid sequence of position
2 to position 195; and [0441] <74> a peptide comprising the
amino acid sequence of position 2 to position 205.
[0442] Further, in the present invention, peptides specified below
can also be included in examples of the peptide having cell
migration-stimulating activity:
[0443] a peptide having cell migration-stimulating activity which
comprises a portion of the amino acid sequence of any one of SEQ ID
NOs: 1, 3, and 5, wherein the peptide meets the following
condition:
[0444] when two peptides are selected from the group of <1>
to <74> above with the short one being A and the long one
being B, the peptide comprises at least A, and consists of the
whole B or a portion thereof.
[0445] Further, the present invention provides peptides comprising
at least any of the amino acid sequences below and having cell
migration-stimulating activity, and uses thereof. Such peptides
also include peptides having cell migration-stimulating activity in
which an amino acid sequence of one or more (for example, 200 or
fewer, 100 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or
fewer, 10 or fewer, 5 or fewer, 3 or fewer, 2 or fewer, without
limitation) amino acids have been added to any of the amino acid
sequences below. The peptide having cell migration-stimulating
activity which comprises at least any one of the amino acid
sequences below does not include peptides consisting of the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5. [0446] [1] the
amino acid sequence of position 17 to position 25 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0447] [2] the
amino acid sequence of position 45 to position 74 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0448] [3] the
amino acid sequence of position 55 to position 84 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0449] [4] the
amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; and [0450] [5]
the amino acid sequence of position 89 to position 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5;
[0451] The amino acid sequence of position 1 to position 85 and the
amino acid sequence of position 86 to position 169 in mouse, rat,
and human HMGB1 are known as A-box and B-box, respectively. The
amino acid sequences of positions 1 to 169 of mouse, rat, and human
are all identical, and maintain 100% identity. Also, the amino acid
sequences of position 14 to position 25 in mouse, rat, and human
HMGB2 are identical to HMGB1.
[0452] The present invention provides peptides having cell
migration-stimulating activity mentioned above. The present
invention further provides DNAs encoding those peptides, vectors
inserted with the DNAs, and transformed cells introduced with the
vectors. DNAs encoding peptides of the present invention, vectors
inserted with the DNAs, and transformed cells introduced with the
vectors are produced using known techniques. The above DNAs may be,
for example, artificially synthesized DNAs (for example, degenerate
mutants) as long as they encode peptides of the present
invention.
[0453] The present invention also provides peptides of the present
invention produced using cells, and peptides of the present
invention synthesized artificially. Peptides of the present
invention can be obtained as recombinants by incorporating a DNA
encoding the peptide into an appropriate expression system, or can
be synthesized artificially. To obtain a peptide of the present
invention by genetic engineering methods, a DNA encoding the
peptide is incorporated into an appropriate expression system and
allowed to express the peptide.
[0454] Thus, the present invention provides a method of producing a
peptide of the present invention, comprising steps (a) and (b)
below: [0455] (a) introducing a DNA encoding a peptide of the
present invention into cells, and expressing said peptide; and
[0456] (b) collecting said peptide from the cells.
[0457] Also, the present invention provides a method of producing a
peptide of the present invention which has higher cell
migration-stimulating activity than an HMGB1 protein, comprising
steps (a) and (b) below: [0458] (a) introducing a DNA encoding a
peptide of the present invention into cells, and expressing said
peptide; and [0459] (b) collecting said peptide from the cells.
[0460] This production method can further comprise the following
step: [0461] (c) selecting said peptide which has higher cell
migration-stimulating activity than the HMGB1 protein.
[0462] Hosts that may be applied in the present invention include,
but are not limited to, prokaryotic cells and eukaryotic cells.
Further, hosts that may be applied in the present invention also
include, but are not limited to, bacteria (for example, E. coli),
yeasts, animal cells (for example, mammalian cells such as HEK293
cell and CHO cell, and insect cells such as silkworm cells), plant
cells, and such.
[0463] Examples of host/vector systems applicable to the present
invention include the expression vector pGEX and E. coli. With
pGEX, foreign genes can be expressed as a fusion protein with
glutathione-S-transferase (GST) (Gene, 67: 31-40, 1988). pGEX
incorporated with DNA encoding a peptide of the present invention
is introduced into an E. coli strain such as BL21 by heat shock,
incubated for an appropriate time and then
isopropylthio-.beta.-D-galactoside (IPTG) is added to induce the
expression of a GST-fused peptide. Since GST of the present
invention adsorbs onto Glutathione Sepharose 4B, the expression
product is readily separated and purified by affinity column
chromatography.
[0464] In addition, the following may also be applied as
host/vector systems to obtain genetic recombinants of the peptides
of the present invention. First, when bacteria are used as hosts,
expression vectors for fusion proteins that utilize tags and the
like are commercially available. The recombinants of the present
invention also include those to which a tag or a partial peptide
thereof is attached.
[0465] Tags attached to the peptides of the present invention are
not particularly limited as long as they do not affect the activity
of the peptides of the present invention. Examples include a
histidine tag (such as 6.times. His or 10.times. His), HA tag, FLAG
tag, GST tag, T7-tag, HSV-tag, E-tag, lck tag, and B-tag.
[0466] Regarding yeasts, yeasts belonging to the genus Pichia are
known to be effective for the expression of sugar chain-containing
proteins. In terms of the addition of sugar chains, expression
systems that utilize baculovirus vector with insect cells as a host
are also useful (Bio/Technology, 6: 47-55, 1988). Further, using
mammalian cells, transfection of a vector is carried out using
promoters such as CMV, RSV, and SV40. Any of these host/vector
systems can be used as an expression system of the peptides of the
present invention. Moreover, genes can also be introduced using
plasmid vectors and viral vectors such as retrovirus vectors,
lentivirus vectors, adenovirus vectors, adeno-associated virus
vectors, Sendai virus vectors, Sendai virus envelope vectors, and
papilloma virus vectors, without limitation thereto. The vectors
may also contain a promoter DNA sequence which effectively induces
gene expression, a factor which regulates gene expression, and any
molecule necessary for maintaining the stability of DNA.
[0467] Thus obtained proteins of the present invention may be
isolated from inside or outside (medium and such) of the host
cells, and can be purified as proteins that are substantially pure
and homogenous. Proteins may be separated and purified using
separation and purification methods which are commonly used in
protein purification, and are not particularly limited. For
example, proteins can be separated and purified by appropriately
selecting and combining a chromatography columns, filters,
ultrafiltration, salting out, solvent precipitation, solvent
extraction, distillation, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, isoelectric focusing electrophoresis,
dialysis, recrystallization, and the like.
[0468] Examples of chromatographies include affinity
chromatography, ion-exchange chromatography, hydrophobic
chromatography, gel filtration, reverse phase chromatography, and
adsorption chromatography (Marshak et al., Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed
Daniel R. Cold Spring Harbor Laboratory Press, 1996). These
chromatographies can be performed using liquid phase
chromatographies such as HPLC and FPLC.
[0469] Moreover, peptides of the present invention are preferably
substantially purified peptides. Here, the term "substantially
purified" means that the purity of the peptide of the present
invention (proportion of the peptide of the present invention in
total protein components) is 50% or more, 60% or more, 70% or more,
80% or more, 90% or more, 95% or more, 100% or close to 100%. The
upper limit for "close to 100%" depends on the purification
techniques and analytical techniques of those skilled in the art,
of which examples are 99.999%, 99.99%, 99.9%, 99%, and the
like.
[0470] Moreover, a substantially purified protein includes any
protein purified by any purification method as long as the protein
purity is as mentioned above. Examples include, but are not limited
to, proteins substantially purified by appropriately selecting and
combining the above-mentioned chromatography columns, filters,
ultrafiltration, salting out, solvent precipitation, solvent
extraction, distillation, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, isoelectric focusing electrophoresis,
dialysis, recrystallization, and the like.
[0471] In the present invention, cells secreting a peptide of the
present invention can also be produced by the following manner. A
vector is produced by inserting a DNA encoding the peptide linked
with a secretion signal-encoding DNA (for example, ATG GAG ACA GAC
ACA CTC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TCC ACT GGT
GAC; SEQ ID NO: 10) into a known expression vector or a gene
therapy vector. The produced vector is introduced into mammalian
cells such as fibroblasts (such as normal skin fibroblasts and cell
lines derived therefrom), insect cells, and other cells.
[0472] Examples of secretion signal-encoding DNAs include, but are
not limited to, DNAs having the above-described sequence.
Furthermore, there are no particular limitations in the animal
species from which these cells derive, although cells of the target
animal species subjected to vector administration, cells from the
target itself, or cells derived from a blood relative of the target
subjected to vector administration, are preferably used.
[0473] Meanwhile, peptides consisting of a portion of HMGB1 can be
artificially synthesized. For the peptide synthesis methods in the
present invention, peptides can be chemically synthesized by either
a liquid-phase peptide synthesis method or a solid-phase peptide
synthesis method. In the present invention, peptides that are
synthesized using a solid-phase peptide synthetic method are
preferable. Solid-phase peptide synthesis is one of the generally
used methods in chemical peptide synthesis. Polystyrene polymer gel
beads with a diameter of about 0.1 mm, modified with amino groups
on their surface, or such can be used as the solid phase, and an
amino acid chain is extended one by one by dehydration reaction.
When the sequence of a peptide of interest is developed, it is
excised from the solid-phase surface to obtain the substance of
interest. By solid-phase synthesis, it is possible to synthesize
ribosome peptides, which are difficult to be synthesized within
bacteria, to introduce non-natural amino acids such as D-amino
acids or heavy-atom derivatives, to modify peptide and protein
backbones, and such. In solid-phase synthesis, long peptide chains
of 70 to 100 or more amino acids may be synthesized by using native
chemical ligation to bind two peptide chains.
[0474] Methods for administering a composition of the present
invention include oral administration and parenteral
administration. Specifically, parenteral administration includes,
but is not limited to, injection, transnasal administration,
transpulmonary administration, transdermal administration, and
such. As examples of injection, intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, and such can be used to administer a composition of the
present invention systemically or locally (for example, under the
skin, in the skin, on the surface of skin, eyeball or palpebral
conjunctiva, nasal cavity mucosa, intraoral mucosa and mucosa of
the gastrointestinal tract, vaginal mucosa/intrauterine mucosa,
damage site or such).
[0475] Methods of administering a composition of the present
invention include, but are not limited to, for example,
intravascular administration (intra-arterial administration,
intravenous administration, or such), blood administration,
intramuscular administration, subcutaneous administration,
intradermal administration, intraperitoneal administration.
[0476] There is no limitation on the site of administration, and
for example, it may be a tissue site in need of regeneration or its
nearby region, a site different from the tissue in need of
regeneration, or a site distant to and different from the tissue in
need of regeneration. The site is, for example, in the blood (in
arteries, in veins, or such), muscle, under the skin, in the skin,
in the abdominal cavity, or such, without being limited
thereto.
[0477] The method of administration may be appropriately selected
according to the age and the symptoms of the patient. When a
peptide of the present invention is administered, the dose per time
of the protein can be selected within a range of 0.0000001 mg to
1000 mg per kg body weight of a patient. Alternatively, the dose
can be selected within a range of 0.00001 mg to 100000 mg per body
of patient, for example. When administering cells secreting a
peptide of the present invention or gene therapy vectors inserted
with DNA encoding the peptide, they may be administered such that
the amount of the peptide is within the above range. However, the
dosage of pharmaceutical compositions of the present invention is
not limited thereto.
[0478] Pharmaceutical compositions of the present invention can be
formulated according to the usual methods (for example, Remington's
Pharmaceutical Science, latest edition, Mark Publishing Company,
Easton, U.S.A), and may contain pharmaceutically acceptable
carriers and additives together. Examples include surfactants,
excipients, colorants, perfumes, preservatives, stabilizers,
buffers, suspending agents, isotonizing agents, binders,
disintegrants, lubricants, flow promoters, and flavoring agents,
although they are not limited thereto and other common carriers may
be appropriately used. Specific examples include light anhydrous
silicic acid, lactose, crystalline cellulose, mannitol, starch,
carmellose calcium, carmellose sodium, hydroxypropylcellulose,
hydroxypropylmethylcellulose, polyvinylacetaldiethylamino acetate,
polyvinylpyrrolidone, gelatin, medium-chain fatty acid
triglyceride, polyoxyethylene hydrogenated castor oil 60, white
sugar, carboxymethyl cellulose, corn starch, and inorganic
salts.
[0479] The present invention provides a kit comprising a substance
of any one of (a) to (c) below: [0480] (a) a peptide consisting of
a portion of an HMGB1 protein and having cell migration-stimulating
activity; [0481] (b) a cell secreting the peptide of (a); and
[0482] (c) a vector into which a DNA encoding the peptide of (a) is
inserted.
[0483] The kit can be used for stimulating cell migration,
mobilizing bone marrow cells from bone marrow to peripheral blood,
or regenerating tissues. Examples of the kit include those
containing: (1) the above-described substance dissolved in
fibrinogen and (2) thrombin; or (1) the above-described substance,
(2) fibrinogen, and (3) thrombin. In the present invention, it is
possible to use commercially-available fibrinogen and thrombin,
including, for example, fibrinogen HT-Wf (Benesis-Mitsubishi
Pharma), Beriplast (ZLB Behring), Tisseel (Baxter), Bolheal
(KAKETSUKEN), and TachoComb (ZLB Behring); however, they are not
limited to these examples.
[0484] Further, the use of a peptide consisting of a portion of an
HMGB1 protein and having cell migration-stimulating activity, a
cell secreting the peptide, and a vector into which a DNA encoding
the peptide can be expressed as in (1) to (9) below. [0485] (1) A
method of stimulating migration of a cell, which comprises
administering an effective amount of a substance of any one of (a)
to (c) below;
[0486] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0487] (b) a cell secreting the peptide of (a); and
[0488] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0489] (2) A method of mobilizing a cell from bone marrow
to peripheral blood, which comprises administering an effective
amount of a substance of any one of (a) to (c) below:
[0490] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0491] (b) a cell secreting the peptide of (a); and
[0492] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0493] (3) A method of regenerating a tissue, which
comprises administering an effective amount of a substance of any
one of (a) to (c) below:
[0494] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0495] (b) a cell secreting the peptide of (a); and
[0496] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0497] (4) Use of a substance of any one of (a) to (c)
below in the manufacture of a composition used for stimulating
migration of a cell:
[0498] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0499] (b) a cell secreting the peptide of (a); and
[0500] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0501] (5) Use of a substance of any one of (a) to (c)
below in the manufacture of a composition used for mobilizing a
cell from bone marrow to peripheral blood:
[0502] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0503] (b) a cell secreting the peptide of (a); and
[0504] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0505] (6) Use of a substance of any one of (a) to (c)
below in the manufacture of a composition used for regenerating a
tissue:
[0506] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0507] (b) a cell secreting the peptide of (a); and
[0508] (c) a vector into which a DNA encoding the peptide of (a).
[0509] (7) A substance of any one of (a) to (c) below for use in a
method of stimulating migration of a cell:
[0510] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0511] (b) a cell secreting the peptide of (a); and
[0512] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0513] (8) A substance of any one of (a) to (c) below for
use in a method of mobilizing a cell from bone marrow to peripheral
blood:
[0514] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0515] (b) a cell secreting the peptide of (a); and
[0516] (c) a vector into which a DNA encoding the peptide of (a) is
inserted. [0517] (9) A substance of any one of (a) to (c) below for
use in a method of regenerating a tissue:
[0518] (a) a peptide consisting of a portion of an HMGB1 protein
and having cell migration-stimulating activity;
[0519] (b) a cell secreting the peptide of (a); and
[0520] (c) a vector into which a DNA encoding the peptide of (a) is
inserted.
[0521] Also, the use of a peptide comprising at least any of the
amino acid sequences below and having cell migration-stimulating
activity can be rephrased similarly as above. [0522] [1] the amino
acid sequence of position 17 to position 25 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0523] [2] the
amino acid sequence of position 45 to position 74 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0524] [3] the
amino acid sequence of position 55 to position 84 in the amino acid
sequence of any one of SEQ ID NOs: 1, 3, and 5; [0525] [4] the
amino acid sequence of position 85 to position 169 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5; and [0526] [5]
the amino acid sequence of position 89 to position 185 in the amino
acid sequence of any one of SEQ ID NOs: 1, 3, and 5.
[0527] All prior art documents cited herein are incorporated herein
by reference.
[0528] Herein below, the present invention will be further
illustrated with reference to Examples, but it is not to be
construed as being limited thereto.
EXAMPLE 1
Purification of HMGB-1 and HMGB1-Derived Peptides Using HEK293
[0529] RNA was extracted from newborn mouse skin using Trizol
(Invitrogen), and then cDNA was synthesized using SuperScript III
cDNA synthesis kit (Invitrogen). Using this cDNA as a template,
HMGB1 cDNA was amplified by polymerase chain reaction (PCR). The
resulting cDNA was inserted into pCAGGS, a plasmid vector for
protein expression in mammalian cells, such that the vector would
express the protein attached with an IgG .kappa. chain signal
sequence as a secretion signal, and with an HA tag, GST tag, and
6.times. His tag sequences at the N terminus of its amino acid
sequence for the convenience of purification (FIG. 1). In addition,
a sequence cleaved by HRV3C was inserted between the His tag and
the protein or peptide of interest. After digestion with HRV3C, a
peptide fragment of Gly Pro Gly Thy Gln (SEQ ID NO: 7) will be
attached to the N-terminal of the protein or peptide of interest.
In the meantime, restriction sites were added to the cDNA of the
full-length HMGB1 or peptide by PCR, and the cDNA was inserted into
the KpnI/EcoRI site of the vector.
[0530] The pCAGGS expression vector produced above was transfected
into a human fetal kidney cell-derived cultured cell line HEK293
using polyethyleneimine (PEI). After 48 hours, the cells and
culture supernatant were collected. The cells and culture
supernatant were separately collected by centrifugation at 4,400 G
at 4.degree. C. for five minutes. Then, the collected supernatant
was filtered through a cellulose acetate filter having pores with a
diameter of 0.8 .mu.m and then through a nitrocellulose filter
having pores with a diameter of 0.45 .mu.m to prepare a sample
removed of insoluble fractions. The sample was loaded onto 5-ml
HisTrap FF (GE) equilibrated with 50 ml of 50 mM Tris HCl (pH 8.0)
containing 50 mM NaCl, and then the absorbed components were washed
with 50 mM Tris HCl (pH 8.0) containing 50 mM NaCl and 10 mM
imidazole to remove nonspecifically adsorbed components. The
specifically adsorbed components were eluted from the column using
50 mM Tris HCl (pH 8.0) containing 50 mM NaCl and 100 mM imidazole.
The adsorbed fractions were fractionated into silicone-coated
plastic tubes (500 .mu.l/tube). Protein-containing fractions were
combined together, and then imidazole was removed using a desalting
column PD10 (GE). The fractions were eluted using 50 mM Tris HCl
(pH. 7.5) containing 150 mM NaCl. HRV3C (Novagen) was added to the
eluted samples and the mixture was incubated at 4.degree. C. for
eight hours. After cleavage of the tags, the sample was loaded onto
a 1-ml HiTrap Heparin column (GE) equilibrated with 50 mM Tris HCl
(pH 7.5) containing 150 mM NaCl. The inside of the column was
washed with 50 mM Tris HCl (pH 7.5) containing 150 mM NaCl. The
protein or peptide bound to the column was eluted with 50 mM Tris
HCl (pH 7.5) containing 1,000 mM NaCl.
Migration Assay
[0531] Cells of a mouse bone marrow mesenchymal stem cell line
(MSC-1 cells, established by Tamai et al. of Osaka University
(PDGFR.alpha.-positive cells in bone marrow are mobilized by high
mobility group box 1 (HMGB1) to regenerate injured epithelia.
(Tamai et al., Proc Natl Acad Sci USA. Apr. 4, 2011))) were
detached from dishes using trypsin, and collected by centrifugation
at 1200 rpm and 4.degree. C. for 10 minutes. The resulting pellet
was loosened and suspended at a cell concentration of
2.0.times.10.sup.6 to 3.0.times.10.sup.6 cells/ml by adding
Dulbecco's Modified Eagle Medium (D-MEM) containing 10% fetal
bovine serum (FBS). The recombinant protein and peptides produced
in HEK293 were diluted with D-MEM containing 10% FBS. The negative
control used was phosphate buffered saline (PBS). An acrylic Boyden
chamber was used; cells of the mouse bone marrow mesenchymal stem
cell line prepared at a cell concentration of 3.times.10.sup.6
cells/ml were placed in its upper layer, while a diluted protein or
peptide is added to the bottom layer. More specifically, a 28-.mu.l
aliquot of a protein or peptide solution was added to each well of
the bottom plate of a 48-well chemotaxis chamber (NEURO PROBE
48WELL CHEMOTAXIS CHAMBER), and a polycarbonate membrane with
8-.mu.m pores (Neuro Probe, Inc, Cat: 866-417-0014) was placed on
the bottom plate. Then, an upper plate was placed on the membrane
and screwed tightly. 50 .mu.l of cells of the mouse bone marrow
mesenchymal stem cell line after concentration adjustment were
added to the upper plate wells. The chamber was placed in an
incubator under 5% CO.sub.2 at 37.degree. C. After four hours, the
membrane was removed from the chamber and stained with Diff-Quik
(Sysmex, Cat: 16920) to detect cells that migrated through membrane
pores to the lower compartment.
Results
[0532] The whole mouse HMGB1 (1-215), and a peptide of positions 1
to 84 (1-84), a peptide of positions 85 to 169 (85-169), a peptide
of positions 1 to 44 (1-44), a peptide of positions 45 to 84
(45-84), and the negative control (PBS) were assessed for the
presence of migration-promoting activity. All the protein and
peptides were used at 50 .mu.g/ml. The 85-169 did not show
detectable migration-promoting activity, while the remaining 1-215,
1-84, 1-44, and 45-84 showed migration-promoting activity (FIG.
2A).
[0533] Furthermore, a peptide of positions 45 to 215 (45-215) and a
peptide of positions 63 to 215 (63-215) were used at concentrations
of 5, 15, and 25 .mu.g/ml to test the migration-promoting activity
(FIG. 3).
Discussion
[0534] In mouse, rat, and human HMGB1 (SEQ ID NOs: 3, 5, and 1,
respectively), the amino acid sequence spanning positions l to 85
is known as A-box, while the amino acid sequence spanning positions
86 to 169 is known as B-box. Among mouse, rat, and human, the amino
acid sequence of positions 1 to 185 is completely identical,
maintaining 100% identity. The amino acid sequence from positions
186 to 215 is a repeat sequence of glutamic acid and aspartic acid,
which is 100% identical between mouse and rat and only differs in
two amino acids from the human sequence. The migration-promoting
activity of the fragment 85-169 was undetectable, suggesting that
it lacks the activity or has the activity below the detection limit
under the conditions of the present experiment. On the other hand,
1-84, 1-44, and 45-84 showed excellent migration-promoting
activity. Therefore, it is predicted that a domain with
migration-promoting activity exist at least in two locations:
within the amino acid sequence of positions 1 to 44 and the amino
acid sequence of positions 45 to 84. HMGB1 is known to promote the
migration of cells such as dendritic cells, and the migration is
believed to be induced when HMGB1 stimulates a receptor called
RAGE. The RAGE-binding domain is known to be situated in the region
corresponding to amino acid positions 150 to 181 in HMGB1. The
present discovery that at least two domains different from the
RAGE-binding domain promoted the migration of bone marrow
mesenchymal stem cells is surprising.
[0535] Both 45-215 and 63-215 exhibited migration-promoting
activity in a concentration-dependent manner. The activity of
45-215 was stronger as compared to 63-215. It is therefore
predicted that there is at least a domain with migration-promoting
activity within the amino acids from positions 63 to 84.
Furthermore, the following peptides produced using HEK293 also
showed the activity of promoting the migration of bone marrow
mesenchymal stem line MSC-1: [0536] a peptide comprising the amino
acid sequence of positions 1 to 42 (1-42), [0537] a peptide
comprising the amino acid sequence of positions 1 to 43 (1-43),
[0538] a peptide comprising the amino acid sequence of positions 1
to 45 (1-45), [0539] a peptide comprising the amino acid sequence
of positions 1 to 46 (1-46), [0540] a peptide comprising the amino
acid sequence of positions 1 to 47 (1-47), [0541] a peptide
comprising the amino acid sequence of positions 1 to 48 (1-48),
[0542] a peptide comprising the amino acid sequence of positions 1
to 49 (1-49), [0543] a peptide comprising the amino acid sequence
of positions 1 to 50 (1-50), [0544] a peptide comprising the amino
acid sequence of positions 1 to 51 (1-51), [0545] a peptide
comprising the amino acid sequence of positions 1 to 52 (1-52), and
[0546] a peptide comprising the amino acid sequence of positions 1
to 62 (1-62).
EXAMPLE 2
Sorting of Primary Cultured PDGFR.alpha.-Positive Bone Marrow
Mesenchymal Stem Cells and Assessment of Migration-Promoting
Activity
[0547] Thigh and tibial bones were excised from donor mice:
B6.129S4-Pdgfratm11(EGFP)Sor/J (PDGFR.alpha.-GFP Mouse). After
removing attached muscles and other tissues, the bones were crushed
finely and incubated with 0.2% collagenase (Roche, REF:
10103586001)/DMEM/2% FBS(filtrated) at 37.degree. C. for 40
minutes. Then, cell aggregates and muscle tissues were removed by
filtration through a 40-.mu.m nylon mesh. After centrifugation at
1200 rpm for 10 minutes, the resulting cells were suspended in
.alpha.MEM containing 10% FBS and 1% P/S and cultured in an
incubator under 5% CO.sub.2 at 37.degree. C. until they reached
100% confluence. The cells were harvested and the following
experiment was carried out according to the protocol attached to
CD11b MicroBeads (Miltenyi Biotec; order No: 130-049-601). The
cells were adjusted to 10.sup.7 cells/90 .mu.l with PBS(-), and
CD11b MicroBeads were added at 10 .mu.l/10.sup.7 cells. After 15
minutes of reaction at 4.degree. C., the cells were washed twice
and suspended in 500 .mu.l of PBS(-). The tube was placed in
AutoMACS separator and the cells were collected according to the
separation program "Depletes". The collected cells were plated onto
an adherent cell culture dish. After adhesion, GFP fluorescence was
observed using a fluorescence microscope. The above-described
peptide 1-44 produced in HEK293 was tested for the
migration-promoting activity using CD11b-negative cells. The
peptide was used at a concentration of 40 .mu.g/ml.
Results
[0548] PDGFR.alpha.GFP cells were barely detectable among
CD11b-positive cells, while a large number of PDGFR.alpha.GFP cells
were observed among CD11b-negative cells (FIG. 4). Meanwhile,
peptide 1-44 produced using HEK293 exhibited strong
migration-promoting activity on CD11b-negative,
PDGFR.alpha.-positive cells (FIG. 5).
Discussion
[0549] CD11b-negative, PDGFR.alpha.-positive cells are considered
to contain a large number of bone marrow mesenchymal stem cells,
which are a type of bone marrow multipotent stem cells. Peptide
1-44 is expected to show migration-promoting activity not only on
the established line of bone marrow mesenchymal stem cell but also
primary cultured bone marrow mesenchymal stem cells.
Test for the Expression of PDGFR.alpha. Protein in Human Bone
Marrow Mesenchymal Stem Cells
Methods
[0550] Human mesenchymal stem cells (hMSC) (Takara Bio; Product No.
PT034) were cultured using human mesenchymal stem cell
chemically-defined medium kit (MSCGM-CD(tm) BulletKit(tm)) (Takara
Bio; Product No. B0632) according to the product manual. At least
cells that had been passaged five times or less were used in
experiments.
[0551] For Western blotting, about 5.times.10.sup.7 cells were
harvested and suspended in 1 ml of PBS. The cell suspension was
combined with 200 .mu.l of 6.times. SDS-PAGE sample buffer, and
heated at 95.degree. C. for 5 minutes. A bacterial cell lysate of
E. coli (JM109) expressing rat PDGFR.alpha. was dissolved in the
sample buffer and used as a positive control. Then, 20 .mu.l of
each sample and Precision Plus Dual color Standard (Bio-Rad (cat #:
161-0374) as molecular weight markers were electrophoresed on a
7.5% SDS-PAGE gel. After electrophoresis, the gel was saved and
transferred onto a PVDF membrane according to a conventional
method. The sample-transferred PVDF membrane was blocked by
immersing in 2% skim milk/0.1% Tween20 (PBS-T) at room temperature
for one hour. Excess skim milk on the membrane was washed off using
PBS-T. PDGFR.alpha. Rabbit anti-Human Polyclonal antibody (Lifespan
Bioscience (cat #: LS-C9640) as a primary antibody was diluted
3000-fold with PBS-T/2% skim milk, and the blocked membrane was
immersed in it for one hour. Then, the membrane was washed a total
of three times by immersing in PBS-T for 10 minutes. Anti-Rabbit
IgG, HRP-Linked Whole Ab Donkey (GE healthcare (cat #: NA934) as a
secondary antibody was diluted 15000-fold with PBS-T/2% skim milk,
and the membrane was immersed in it for one hour. Then, the
membrane was washed a total of three times by immersing in PBS-T
for 10 minutes. PDGFR.alpha. bands were detected using ECL Prime
(GE healthcare (cat #: RPN2232) according to the product
manual.
Results
[0552] The positive control, rat PDGFR.alpha. produced in E. coli,
was detected as a band with a size of about 170 kDa. Meanwhile,
PDGFR.alpha. of human bone marrow mesenchymal stem cells was
detected at a position of a little higher molecular weight (FIG.
2B).
Discussion
[0553] PDGFR.alpha. protein expression was detected by Western
blotting not only in mouse but also in human bone marrow
mesenchymal stem cells. The reason why the size was a little larger
than that of rat PDGFR.alpha. produced in E. coli may be because of
the presence of modification such as glycosylation. PDGFR.alpha.
was demonstrated to be also expressed in human bone marrow
mesenchymal stem cells.
EXAMPLE 3
Assessment of Primary Cultured PDGFR.alpha.-Positive Bone Marrow
Mesenchymal Stem Cells for Multipotency
[0554] FACS Sorting of PDGFR.alpha.-Positive, Lineage-Negative,
c-Kit-Negative Cells
[0555] Under sufficiently deep anesthesia with isoflurane, C57B16
mice (male, 6 weeks old) were euthanized by carbon dioxide
inhalation. Thigh and tibial bones were excised and fat and muscle
tissues were removed from them. The bones were soaked in EtOH to
thoroughly remove attached tissues from them. Bone marrow tissues
were obtained using a syringe with 26G needle. The obtained bone
marrow cells were combined with DMEM containing 0.2% Collagenase A
and incubated at 37.degree. C. for 40 minutes. After adding DMEM
containing 10% FBS, the cells were centrifuged at 1500 rpm for 10
minutes. The supernatant was discarded and the precipitated bone
marrow cells were collected.
[0556] The cells were plated in a culture dish with a diameter of
10 cm and cultured using D-MEM containing 10% FBS supplemented with
1.times. streptomycin-penicillin in an incubator under 5% CO.sub.2
at 37.degree. C. The medium was changed with fresh one every three
days. The medium of the adherent cells were discarded and the cells
were washed twice by adding 10 ml of PBS. After adding 5 ml of
0.25% trypsin, the cells were incubated at 37.degree. C. for 10
minutes. The cells detached from the culture dish were harvested
and D-MEM containing 10% FBS was added to stop the reaction of
trypsin. The cells were centrifuged at 1200 rpm for 3 minutes. The
precipitated cells were collected, suspended at 1.times.10.sup.6
cells/100 .mu.l in PBS containing 2% FBS, and dispensed into each
well of a round-bottomed 96-well plate. APC-mouse Lineage antibody
cocktail (BD Phamingen; Cat. 558074) as a primary antibody was
added in 10 .mu.l/well. 1 .mu.l each of PE-mouse CD140a
(PDGFR.alpha.) (BD Bioscience; Cat. 12-1401-81) and FITC-mouse
c-kit (BD Bioscience) were added to each well. The cells were
incubated at 4.degree. C. in the dark for 20 minutes. After adding
200 .mu.l of PBS containing 2% FBS to each well, the cells were
centrifuged at 1500 rpm for 10 minutes. The supernatant was
discarded. Then, the cells were washed twice in the same manner.
The cells were sorted using the BD FACSAria cell sorter.
Induction of Osteogenic Differentiation
[0557] When cells were grown to 70% confluency, the medium was
changed with an osteogenic differentiation medium (R & D;
prepared with SC010) every two to three days. The cells were
cultured in an incubator at 37.degree. C. under 5% CO.sub.2. The
culture was continued for about three weeks.
ALP Staining
[0558] Cells subjected to induction of osteogenic differentiation
were fixed for 10 seconds with the fixative solution of the kit
(prepared in advance from the fixative preparation solution), and
stained at 37.degree. C. for three minutes with a substrate
solution prepared from Fast Blue RR Salt and the substrate stock
solution (Muto Pure Chemicals; Cat. No. 1568-2). ALP-positive cells
were stained bluish purple.
Induction of Adipogenic Differentiation
[0559] When cells were grown to 100% confluency, the medium was
changed with an adipogenic differentiation medium (R & D;
prepared with SC010) every three to four days. The cells were
cultured in an incubator at 37.degree. C. under 5% CO.sub.2. The
culture was continued for about two weeks.
Oil Red Staining
[0560] Cells subjected to induction of adipogenic differentiation
were fixed with the propylene glycol fixative solution attached to
the kit, and then adipocytes were stained using Oil Red 0 Solution
(DBS; item #KT 025).
Results
[0561] Cells stained bluish purple were observed among the cells
subjected to induction of osteogenic differentiation, indicating
that the cells differentiated into osteoblast cells (FIG. 6A).
Meanwhile, adipocytes containing oil drops stained red were
observed among the cells subjected to induction of adipogenic
differentiation, indicating the cells differentiated into
adipocytes (FIG. 6B).
Discussion
[0562] Bone-marrow PDGFR.alpha.-positive cells are expected to at
least contain bone marrow mesenchymal stem cells capable of
osteogenic and adipogenic differentiation.
EXAMPLE 4
Assessment of Synthetic Peptides for Migration-Promoting
Activity
[0563] The peptides listed below were custom synthesized using the
solid phase method by Medical & Biological Laboratories (MBL).
The peptides were synthesized based on the mouse HMGB1 sequence
(SEQ ID NO: 3). Synthetic peptides described in subsequent Examples
were also prepared based on the mouse HMGB1 sequence. [0564] A
synthetic peptide consisting of the amino acid sequence from
positions 1 to 10 of HMGB1 (1-10); [0565] a synthetic peptide
consisting of the amino acid sequence from positions 1 to 34 of
HMGB1 (1-34); [0566] a synthetic peptide consisting of the amino
acid sequence from positions 37 to 62 of HMGB1 (37-62); [0567] a
synthetic peptide consisting of the amino acid sequence from
positions 27 to 62 of HMGB1 (27-62); [0568] a synthetic peptide
consisting of the amino acid sequence from positions 56 to 72 of
HMGB1 (56-72); [0569] a synthetic peptide consisting of the amino
acid sequence from positions 11 to 20 of HMGB1 (11-20); [0570] a
synthetic peptide consisting of the amino acid sequence from
positions 11 to 25 of HMGB1 (11-25); [0571] a synthetic peptide
consisting of the amino acid sequence from positions 11 to 30 of
HMGB1 (11-30); [0572] a synthetic peptide consisting of the amino
acid sequence from positions 11 to 34 of HMGB1 (11-34); [0573] a
synthetic peptide consisting of the amino acid sequence from
positions 11 to 44 of HMGB1 (11-44); [0574] a synthetic peptide
consisting of the amino acid sequence from positions 17 to 44 of
HMGB1 (17-44); [0575] a synthetic peptide consisting of the amino
acid sequence from positions 1 to 25 of HMGB1 (1-25); and [0576]
the whole mouse HMGB1 produced in HEK293 (1-215(HEK)) as a positive
control were adjusted to 100 .mu.g/ml and placed in the lower layer
of a chemotaxis chamber to assess the migration-promoting activity
on bone marrow mesenchymal stem cell line (MSC-1).
Results
[0577] At least synthetic peptides (11-34), (1-34), (11-44),
(1-44), and (11-30) were found to have the activity comparable to
or higher than that of the positive control (FIG. 7). Furthermore,
synthetic peptides (11-25) and (1-25) were also found to have the
activity (FIG. 7).
Discussion
[0578] It is predicted from the results of Example 1 that the amino
acid sequences of positions 1 to 44 and positions 45 to 84 each
contain at least one region having the migration-promoting
activity. From the results of the present experiment of Example 4,
it is predicted that synthetic peptide (11-34) has strong
migration-promoting activity and an active center exists at least
in the amino acid sequence from positions 11 to 34. Furthermore,
synthetic peptide (11-25) was also found to have the activity
although it was slightly weaker. It is expected that there is an
active center region within the amino acid sequence from positions
11 to 25, and the amino acid sequences located before and after the
center enhance the activity.
EXAMPLE 5
Methods
[0579] In order to narrow down the location of the active center,
the shorter peptides listed below were synthesized. [0580] A
synthetic peptide consisting of the amino acid sequence from
positions 11 to 27 of HMGB1 (11-27), [0581] a synthetic peptide
consisting of the amino acid sequence from positions 11 to 28 of
HMGB1 (11-28), [0582] a synthetic peptide consisting of the amino
acid sequence from positions 11 to 29 of HMGB1 (11-29), [0583] a
synthetic peptide consisting of the amino acid sequence from
positions 12 to 30 of HMGB1 (12-30), [0584] a synthetic peptide
consisting of the amino acid sequence from positions 13 to 30 of
HMGB1 (13-30), [0585] a synthetic peptide consisting of the amino
acid sequence from positions 14 to 30 of HMGB1 (14-30), [0586] a
synthetic peptide consisting of the amino acid sequence from
positions 15 to 30 of HMGB1 (15-30), [0587] a synthetic peptide
consisting of the amino acid sequence from positions 16 to 30 of
HMGB1 (16-30), [0588] a synthetic peptide consisting of the amino
acid sequence from positions 17 to 30 of HMGB1 (17-30), [0589] a
synthetic peptide consisting of the amino acid sequence from
positions 18 to 30 of HMGB1 (18-30), [0590] a synthetic peptide
consisting of the amino acid sequence from positions 19 to 30 of
HMGB1 (19-30), [0591] a synthetic peptide consisting of the amino
acid sequence from positions 20 to 30 of HMGB1 (20-30), [0592] a
synthetic peptide consisting of the amino acid sequence from
positions 21 to 30 of HMGB1 (21-30), [0593] a synthetic peptide
consisting of the amino acid sequence from positions 10 to 25 of
HMGB1 (10-25), [0594] a synthetic peptide consisting of the amino
acid sequence from positions 11 to 25 of HMGB1 (11-25), [0595] a
synthetic peptide consisting of the amino acid sequence from
positions 12 to 25 of HMGB1 (12-25), [0596] a synthetic peptide
consisting of the amino acid sequence from positions 13 to 25 of
HMGB1 (13-25), [0597] a synthetic peptide consisting of the amino
acid sequence from positions 14 to 25 of HMGB1 (14-25), [0598] a
synthetic peptide consisting of the amino acid sequence from
positions 15 to 25 of HMGB1 (15-25), [0599] a synthetic peptide
consisting of the amino acid sequence from positions 16 to 25 of
HMGB1 (16-25), [0600] a synthetic peptide consisting of the amino
acid sequence from positions 17 to 25 of HMGB1 (17-25), and [0601]
a synthetic peptide consisting of the amino acid sequence from
positions 186 to 215 of HMGB1 (186-215).
[0602] As positive controls, centrifuged supernatant of the skin of
a one-day-old mouse (one individual) incubated in PBS at 4.degree.
C. for 12 hours, and the whole mouse HMGB1 (HMGB1(HEK_1-215))
produced in HEK293 were used. Cells of a bone marrow mesenchymal
stem cell line (MSC-1) were placed in the upper layer of a
chemotaxis chamber, and the protein and synthetic peptides were
added at a concentration of 5 .mu.M or 10 .mu.M to the lower layer
of the chemotaxis chamber. The migration assay was carried out by
the same method as described in Example 1.
Results
[0603] At least synthetic peptides (11-27), (11-28), (11-29),
(12-30), (13-30), (14-30), and (10-25) exhibited strong
migration-promoting activity at 5 .mu.M. Meanwhile, synthetic
peptides (11-25), (12-25), (13-25), (14-25), (15-25), (15-30),
(16-25), (16-30), (17-25), and (17-30) showed weak activity (FIGS.
8A and 8B).
Discussion
[0604] It is predicted from the results of Example 4 that a domain
having migration-promoting activity is located within the amino
acid sequence from positions 11 to 25. Thus, one of the domains
having migration-promoting activity is expected to be present in
the amino acids from positions 17 to 25 (9 amino acids).
Comparison of HMGB1 Fragments for Migration-Promoting Activity on
Bone Marrow Mesenchymal Stem Cells
Methods
[0605] Each of synthetic peptides 15-30, 16-30, 17-30, 17-44,
45-74, and 55-84, which consists of an HMGB1 fragment, was compared
to a negative control (PBS) for the level of the
migration-promoting activity on bone marrow mesenchymal stem cells
(MSC-1). The Boyden chamber method was used in the same manner as
described above. Each peptide was added at 10 .mu.M to the lower
layer of the chamber. 1.5.times.10.sup.6 cells dispersed in 1 ml of
DMEM containing 10% FBS were placed in the upper layer of the
chamber. A polycarbonate membrane having pores with a diameter of 8
.mu.m was inserted between the upper and lower layers. After four
hours of incubation in an incubator under 5% CO.sub.2 at 37.degree.
C., the membrane was removed and treated with Diff-Quik stain.TM.
to stain only cells that migrated to the lower layer. After
staining, the cells were air-dried, and cells that migrated to the
lower layer were counted under a microscope. The average number was
calculated.
Results
[0606] All the peptides exhibited stronger migration-promoting
activity than that of the negative control.
Discussion
[0607] Peptide synthesis is a highly excellent production method
for pharmaceutical production, because, as compared to production
methods using HEK293 or bacteria such as E. coli, it can ensure a
consistent production amount at low costs and prevent contamination
with biologically-derived toxins and such. On the other hand,
unlike the production in organisms, post-translational modification
and folding do not occur properly, and therefore low molecular
weight peptides containing highly hydrophobic amino acids often
become very insoluble in aqueous solutions. In the present Example,
since the migration-promoting activity of peptides was relatively
weak, the activity strength as compared to the negative control was
measured correctly using a microscope. A strong migration-promoting
activity as compared to the negative control was detected for all
the peptides (FIG. 8C).
EXAMPLE 6
Assessment of Synthetic Peptides for Migration-Promoting
Activity
[0608] Synthetic peptides (1-44) and (1-34) used in Example 4,
peptide (45-74) consisting of the amino acids of positions 45 to
74, and peptide (55-84) consisting of the amino acids of positions
55 to 84 were tested by migration assay using a chemotaxis chamber
in the same manner as described in Example 5. The assay was carried
out simultaneously at two concentrations of 10 .mu.M and 5
.mu.M.
Results
[0609] Both synthetic peptides (45-74) and (55-84) exhibited
migration-promoting activity which was however weaker as compared
to synthetic peptides (1-44) and (1-34) (FIG. 9).
Discussion
[0610] The results of Example 1 showed that peptides (1-84),
(1-44), and (45-84) produced in HEK293 had strong
migration-promoting activity on PDGFR.alpha.-positive mesenchymal
stem cells. The results of Example 4 showed that synthetic peptides
(1-44) and (1-34) retained the strong activity. Meanwhile, the
results of Example 6 here revealed that synthetic peptides (45-74)
and (55-84) also exhibited migration-promoting activity although it
was slightly weaker.
[0611] It is known that peptides and proteins are subjected to
modification such as glycosylation when synthesized in eukaryotic
cells such as HEK293. On the other hand, synthetic peptides do not
undergo modification. The fact that the migration-promoting
activity of peptide (45-84) produced in HEK293 was greater as
compared to synthetic peptides "(45-74) and (55-84)" consisting of
the amino acid sequence of positions 45 to 84 suggests that the
peptide was subjected to certain modification.
[0612] Meanwhile, a past experiment using mesoangioblasts (Palumbo
et al., J. Cell Biol., 164: 441-449, 2004) has shown that the
sequence consisting of the amino acids of positions 1 to 187
resulting from cleavage at the C terminal end retains the cell
migration-promoting activity of HMGB1 (215 amino acids in entire
length) while the sequence consisting of the amino acids of
positions 1 to 89, the sequence consisting of the amino acids of
positions 90 to 176, and the sequence consisting of the amino acids
of positions 1 to 176 have almost no activity. On the other hand,
the portion predicted to be a ligand for RAGE, which is one of
known HMGB1 receptors, corresponds to the sequence consisting of
the amino acids of positions 150 to 181. The document described
above also shows that the migration-promoting activity is
suppressed by a dominant negative of RAGE. Moreover, another report
(Yang et al., J Leukoc Biol. January; 81(1): 59-66, 2007) shows
that the RAGE receptor is also utilized when HMGB1 promotes
migration of dendritic cells. Thus, for the migration-promoting
activity of HMGB1, attention has previously been drawn to the
C-terminal peptide of HMGB1, which is a ligand portion for
RAGE.
[0613] The present Examples succeeded in identifying two regions
having cell migration-promoting activity within the N-terminal
peptide, which had been believed not to have the cell
migration-promoting activity. Excessive inflammation is known to be
an inhibitory factor in tissue regeneration. Since the active
regions discovered in the present Examples are completely different
from the ligand for RAGE, it is expected that they will enable
recruitment of PDGFR.alpha.-positive stem cells while avoiding
recruitment of inflammatory cells such as dendritic cells, and
therefore development of pharmaceuticals with much less side
effects will be possible.
EXAMPLE 7
Quantitative Comparison of Peptide (1-44) and the Whole HMGB1 for
Migration-Promoting Activity
[0614] HEK293 cells were transfected with the expression vector for
mouse HMGB1 (1-44) using polyethyleneimine, and HMGB1 secreted into
cell supernatant was collected and purified (HEK293 transient) in
the same manner as described in Example 1. Also, after transfection
by the same procedure, 2 .mu.g/ml puromycin was added to the
culture medium, and cells constantly secreting HMGB1 (1-44) were
selected by the drug. HMGB1 secreted to this cell supernatant was
purified (HMGB1-stable).
Production of HMGB1-Derived Peptides Using E. Coli
[0615] In order to produce the peptide of amino acids 1-44 using E.
coli, a cDNA for expression of a chimeric peptide to be added to
the N terminal side of cDNA encoding the amino acids of positions 1
to 44 of mouse HMGB1 to be cleaved off by HRV3C was inserted into
pENTR vector (Invitrogen). LR reaction was carried out for transfer
into pDEST17 vector. The expression vector has a T7 promoter and is
capable of protein expression in E. coli. Furthermore, it adds
6.times. His tag to the N terminal end. As a result of HRV3C
cleavage, the 6.times. His tag is removed and a peptide fragment of
Gly Pro Gly Thy Gln (SEQ ID NO: 7) is added to the N terminal end
of the peptide.
[0616] E. coli BL21 (DE3) was transformed with the above-described
expression vector by electroporation. After adding SOC medium, the
E. coli was cultured in a shaker at 37.degree. C. for 60 minutes.
The cells were plated on an LB agar plate containing carbenicillin,
and incubated at 37.degree. C. for 18 hours. Single colonies were
collected and combined with LB containing carbenicillin, and
cultured in a shaker at 37.degree. C. When the O.D. 600 reached
0.4-0.5, IPTG was added at a final concentration of 0.1 mM, and
shaken at 30.degree. C. After 6 hours, the E. coli was harvested,
and centrifuged at 3500 rpm for 30 minutes. The precipitated E.
coli was collected, and 50 mM Tris-HCl (pH 8.0) containing 50 mM
NaCl and 6M urea was added thereto. The E. coli was lysed by
pipetting and loaded onto 5 ml HisTrap FF(GE) equilibrated with 50
mM Tris-HCl (pH 8.0) containing 50 mM NaCl and 6 M urea. Then, the
adsorbed material was washed with 50 mM Tris HCl (pH 8.0)
containing 50 mM NaCl, 6 M urea, and 10 mM imidazole to remove
non-specifically adsorbed components. The specifically adsorbed
material was eluted from the column with 50 mM Tris HCl (pH 8.0)
containing 50 mM NaCl, 6 M urea, and 300 mM imidazole. The adsorbed
material was fractionated into 500 .mu.l fractions in
silicone-coated plastic tubes, and protein-containing fractions
were combined together. Then, imidazole was removed using desalting
column PD10 (GE) and elution was carried out using 50 mM Tris-HCl
(pH 7.5) containing 150 mM NaCl. HRV3C (Novagen) was added to the
eluted sample and allowed to react at 4.degree. C. for 8 hours.
After tag cleavage, the sample was loaded onto a 1-ml HisTrap FF
column equilibrated with 50 mM Tris HCl (pH 7.5) containing 150 mM
NaCl, and the peptide was collected as an unbound fraction.
[0617] The synthetic peptide (1-44) was prepared in the same manner
as described above. The peptides and protein were used at a
concentration of 2 .mu.M for migration assay using a bone marrow
mesenchymal stem cell line (MSC-1). The pore area of the chemotaxis
chamber and the area of migrated cells were measured using image
analysis software.
Results
[0618] When compared per equal mole, peptide (1-44) produced in
HEK293 and peptide (1-44) produced in E. coli both exhibited
migration-promoting activity about 1.6 times greater than the
full-length HMGB1. When compared per equal mass, peptide (1-44)
produced in HEK293 and peptide (1-44) produced in E. coli both
exhibited migration-promoting activity about 8 times greater than
the full-length HMGB1. Meanwhile, the migration-promoting activity
of synthetic peptide (1-44) was 0.57 times greater and 2.86 times
greater than the full-length HMGB1 when compared per equal mole and
per equal mass, respectively (FIG. 10).
Discussion
[0619] The results of Examples 1 and 6 and others suggest that
there are at least one or more active center sites for the
migration-promoting activity in each of the amino acid sequence of
1 to 44 and the amino acid sequence of positions 45 to 84, and thus
there are a total of two or more active center sites. Furthermore,
the results of the present Example 7 demonstrated that peptide
(1-44) produced in HEK293 had migration-promoting activity nearly 8
times that of the full-length HMGB1 when compared per equal mass
(about 1.6 times when compared per equal number of moles). In
addition, peptide (45-84) produced in HEK293 also has comparable
migration-promoting activity (FIG. 2A). The findings described
above demonstrate that HMGB1 has at least two regions that have
greater migration-promoting activity than the same number of moles
of the full-length HMGB1, which are located within the amino acid
sequence of positions 1 to 44 and the amino acid sequence of
positions 45 to 84, and the activity of the full-length HMGB1 is
significantly lower than the sum of the activity of the two
regions. Since the peptide of positions 1 to 44 and the peptide of
positions 45 to 84 were adjacent to each other according to the
result of crystallographic analysis of the full length HMGB1, it is
predicted that these regions inhibit the activity of each other. It
is suggested that the separation into peptides resulted in
elimination of the inhibition and an increase in the activity of
each.
EXAMPLE 8
FACS Analysis of Bone Marrow Mesenchymal Stem Cell Line (MSC-1) for
Expression of PDGFR.alpha., Lineage Marker, and CD44
[0620] Cells of mouse-derived bone marrow mesenchymal stem cell
line MSC-1 were plated in a culture dish with a diameter of 10 cm
and cultured in an incubator under 5% CO.sub.2 at 37.degree. C.
using D-MEM containing 10% FBS supplemented with 1.times.
streptomycin-penicillin. After the cells were grown to 80-90%
confluency, the medium was discarded and the cells were washed
twice by adding 10 ml of PBS. Then, 5 ml of 0.25% trypsin was added
and incubated at 37.degree. C. for 10 minutes. The cells detached
from the culture dish were harvested and DMEM containing 10% FBS
was added to stop the reaction of trypsin. The cells were
centrifuged at 1200 rpm for 3 minutes. The precipitated cells were
collected, and suspended at 1.times.10.sup.6 cells/100 .mu.l of PBS
containing 2% FBS. The cells were dispensed into each well of a
round-bottomed 96-well plate. APC-mouse Lineage antibody cocktail
(BD Phamingen; Cat. 558074) as a primary antibody was added in 10
.mu.l/well. 1 .mu.l each of PE-mouse CD140a (PDGFR.alpha.) (BD
Bioscience; Cat. 12-1401-81) and FITC-mouse CD44 (BD Bioscience;
Cat. 553-133) were added to each well. The cells were incubated at
4.degree. C. in the dark for 20 minutes. A 200-.mu.l aliquot of PBS
containing 2% FBS was added to each well. The cells were
centrifuged at 1500 rpm for 10 minutes. The resulting supernatant
was discarded. Then, the cells were washed twice in the same
manner. The cells were suspended in 100 .mu.l of PBS and analyzed
with BD FACSCant.TM. II.
Results
[0621] Bone marrow mesenchymal stem cells (MSC-1) were
PDGFR.alpha.-positive, Lineage-negative, and CD44-positive (FIG.
11).
Discussion
[0622] The cells used for the migration-promoting activity retain
the properties of PDGFR.alpha.-positive bone marrow mesenchymal
stem cells.
EXAMPLE 9
Migration-Promoting Activity of Synthetic Peptide (1-34) on Mouse
Keratinocytes
[0623] Newborn C57/B16 mice were euthanized using isoflurane and
carbon dioxide inhalation. Then, the mice were washed thoroughly
with EtOH and PBS. The skin together with dermis was exfoliated and
the blood was washed off with PBS. The exfoliated skin was placed
in Dispase I (Sanko Junyaku Co., Cat: GD81060) at 4.degree. C. for
16 hours. The epidermis and dermis were detached with forceps, and
the epidermis was placed in trypsin (Nacalai tesque, Cat: 3554-64)
at 37.degree. C. for 10 minutes. When white turbidity began to
appear, the reaction was stopped with S-MEM (GIBCO, Cat: 11380)/15%
FBS(Ca-)P/S. The resulting cells were centrifuged at 160.times.G
for 5 minutes, and suspended in CnT-07 medium (CELLnTEC, Cat:
CnT-07 BM) and plated in a 10-cm dish. The cells were cultured
under 5% CO.sub.2 at 37.degree. C. and the medium was changed every
three days. The cells were passaged when they reached 80% to 90%
confluency. The cells were harvested from the dish using trypsin.
After inactivation of trypsin with DMEM containing 10% FBS, a
synthetic peptide consisting of the amino acid sequence of
positions 1 to 34 (1-34) was examined for migration-promoting
activity according to the migration assay method described
above.
PDGFR.alpha. Expression in Mouse Keratinocytes
[0624] Newborn 136.129S4-Pdgfratm11(EGFP)Sor/J mice were fixed by
perfusion with 4% PFA. A 1.0.times.1.0 cm.sup.2 area of the newborn
skin was excised, and further fixed by immersion with 4% PFA for 12
hours and then with 30% sucrose for 12 hours at 4.degree. C. After
washing with PBS(-), the skin was cryoembedded in OTC compound and
sliced into 8-.mu.m cryosections in a cryostat. The sections were
washed twice with PBS to wash out the compound, and blocked with
10% goat serum in PBS at room temperature for 1 hour. The primary
antibody used was rabbit anti-keratin 5 (Covance, Cat: PRB-160P) or
rabbit anti-vimentin (Abeam, Cat: ab7783-500) diluted 500 times
with PBS containing 10% goat serum. After five hours of incubation
with the primary antibody at 4.degree. C., the sections were washed
twice with PBS. The secondary antibody used was Alexa Fluor 546
goat anti-rabbit IgG(H+L) (Invitrogen, Cat: A11035) diluted 500
times with PBS containing 10% goat serum. After 45 minutes of
incubation with the secondary antibody at room temperature, the
sections were washed twice with PBS, and incubated with 2 .mu.g/ml
DAPI (4',6-diamino-2-phenylindole) at room temperature for 3
minutes. Then, after washing twice with PBS, the sections were
mounted with a mounting medium containing a fluorescence
antifade.
Results
[0625] Synthetic peptide (1-34) did not show migration-promoting
activity on mouse keratinocytes (FIG. 12A). Furthermore, GFP
fluorescence was not observed on cells positive for keratin 5,
which is a mouse keratinocyte marker (FIG. 12B).
Discussion
[0626] Keratinocytes did not express PDGFR.alpha.. Furthermore,
synthetic peptide (1-34) did not have migration-promoting activity
on keratinocytes.
EXAMPLE 10
Migration-Promoting Activity of Synthetic Peptide (1-34) on Mouse
Skin Fibroblasts
[0627] Newborn C57/B16 mice were euthanized using isoflurane and
carbon dioxide inhalation. Then, the mice were washed thoroughly
with EtOH and PBS. The skin together with dermis was exfoliated and
the blood was washed off with PBS. The exfoliated skin was cut into
fine pieces with scissors. The skin pieces were placed in DMEM
(Nacalai tesque, Cat: 08458-45) containing 0.2% collagenase (Roche,
REF: 10103586001) and shaken at 37.degree. C. for 30 minutes. The
reaction was stopped with DMEM/30% FBS/P/S. The resulting cells
were centrifuged at 160.times.G for 5 minutes and plated in a 10-cm
dish. The cells were cultured under 5% CO.sub.2 at 37.degree. C.
and the medium was changed every three days. The cells were
passaged when they reached 80% to 90% confluency. The cells were
harvested from the dish using trypsin. After inactivation of
trypsin with D-MEM containing 10% FBS, a synthetic peptide
consisting of the amino acid sequence of positions 1 to 34 (1-34)
was assessed for migration-promoting activity according to the
migration assay method described above.
[0628] Newborn B6.129S4-Pdgfratm11(EGFP)Sor/J mice were fixed by
perfusion using 4% PFA. A 1.0.times.1.0 cm.sup.2 area of the
newborn skin was excised, and further fixed by immersion with 4%
PFA for 12 hours and then with 30% sucrose at 4.degree. C. After
washing with PBS(-), the skin was cryoembedded in OTC compound and
sliced into 8-.mu.m cryosections in a cryostat. The sections were
washed twice with PBS to wash out the compound, and blocked with
10% goat serum in PBS at room temperature for 1 hour. The primary
antibody used was rabbit anti-vimentin (Abeam; Cat. ab7783-500)
diluted 500 times with PBS containing 10% goat serum. After five
hours of incubation with the primary antibody at 4.degree. C., the
sections were washed twice with PBS. The secondary antibody used
was Alexa Fluor 546 goat anti-rabbit IgG(H+L) (Invitrogen; Cat.
A11035) diluted 500 times with PBS containing 10% goat serum. After
45 minutes of incubation with the secondary antibody at room
temperature, the sections were washed twice with PBS, and incubated
with 2 .mu.g/ml DAPI (4',6-diamino-2-phenylindole) at room
temperature for 3 minutes. Then, after washing twice with PBS, the
sections were mounted with a mounting medium containing a
fluorescence antifade.
Results
[0629] Synthetic peptide (1-34) exhibited migration-promoting
activity on skin fibroblasts (FIG. 13A). Furthermore, GFP
fluorescence-positive cells were observed among cells positive for
vimentin, which is a fibroblast marker (FIG. 13B).
Discussion
[0630] Skin fibroblasts expressed PDGFR.alpha.. Synthetic peptide
(1-34) had migration-promoting activity on skin fibroblasts. Both
bone marrow mesenchymal stem cells and newborn skin fibroblasts are
positive for PDGFR.alpha.. Peptide (1-34) showed
migration-promoting activity on both cells but not on
keratinocytes, which are PDGFR.alpha.-negative cells. PDGFR.alpha.
is expected to be useful as a marker for cells on which amino acid
sequences comprising peptide (1-34) exhibit migration-promoting
activity.
EXAMPLE 11
Assessment of PDGFR.alpha. Expression in Mouse Skin Fibroblasts
Using FACS
[0631] Newborn B6.12954-Pdgfratm11(EGFP)Sor/J mice were washed
thoroughly with EtOH and PBS. The skin was detached from muscles
and cut into small pieces with a width of 3 mm. The skin pieces
were transferred into DMEM/5% FBS containing 500 unit/ml dispase
and incubated at 4.degree. C. for 18 hours. The dermis was
exfoliated from the epidermis and cut into fine pieces with
scissors. The fine pieces of dermis were placed in DMEM containing
0.2% collagenase and shaken at 37.degree. C. for 30 minutes. After
adding DMEM containing 30% FBS, the resulting cells were
centrifuged at 160.times.G for 5 minutes. The precipitated cells
were plated in a 10-cm dish, and cultured under 5% CO.sub.2 at
37.degree. C. The medium was changed every three days and the cells
were passaged when they reached 80% to 90% confluency. The cells
were harvested from the dish using trypsin, and after adding D-MEM
containing 10% FBS the cells were collected by centrifugation. GFP
fluorescence of cells was detected and analyzed with BD
FACSCant.TM. II.
Results
[0632] 98% or more of fibroblasts in newborn mouse skin were
positive for PDGFR.alpha. (FIG. 14).
Discussion
[0633] PDGFR.alpha.-positive cells were quantified using FACS.
Almost all fibroblasts were shown to be PDGFR.alpha.-positive
cells, as in the result of immunohistochemistry.
EXAMPLE 12
Methods
[0634] 10 .mu.g of synthetic peptide (amino acids of positions 1 to
44) was dissolved in 200 .mu.l of PBS and administered to C57B16
mice (female, 8 weeks old) via the caudal vein using a syringe with
30-gauge needle. An equal volume of PBS was administered as a
negative control. After 12 hours, peripheral blood was collected
from the left ventricle of the heart under systemic anesthesia with
isoflurane. After adding 3 ml of PBS (Nacalai tesque; Cat.
14249-95), 3 ml of Ficoll-Paque Plus (GE Healthcare; Cat.
17-1440-02) was overlaid onto the blood. The blood was centrifuged
in a centrifuge at 400 G and 25.degree. C. for 45 minutes. The
upper serum layer was discarded, and only the cells seen as a white
band in the intermediate layer were collected. Then, 45 ml of PBS
was added to the collected cells, and centrifuged in a centrifuge
at 800 G and 25.degree. C. for 20 minutes. After the supernatant
was discarded, 10 ml of PBS was added and centrifuged at 1500 rpm
and 25.degree. C. for 10 minutes. The supernatant was discarded and
1 ml of hemolysis buffer (HLB; Immuno-Biological Laboratories) was
added to the cells. After pipetting, the cells were allowed to
stand for 5 minutes. Then, 10 ml of PBS was added to the cells and
centrifuged at 1500 rpm and 25.degree. C. for 10 minutes. The
precipitated mononuclear cells were collected and adjusted to
1.times.10.sup.6 cells/100 .mu.l (PBS containing 2% FBS) in a
round-bottomed 96-well plate. 1 .mu.l each of PE-mouse CD140a
(PDGFR.alpha.) (BD Bioscience; Cat. 12-1401-81) or FITC-mouse CD44
(BD Bioscience, Cat.553-133) was added to each well containing
mononuclear cells. The cells were incubated at 4.degree. C. in the
dark for 20 minutes. 200 .mu.l of PBS was added to each well, and
centrifuged at 1500 rpm and 4.degree. C. for 10 minutes. The
supernatant was discarded and again 200 .mu.l of PBS was added to
each well. The cells were centrifuged at 1500 rpm and 4.degree. C.
for 10 minutes. The cells were suspended in 100 .mu.l of PBS, and
300 .mu.l of 1% paraformaldehyde was added thereto. A control was
prepared using an isotype control antibody in the same manner as
described above. The cells prepared as described above were
analyzed using FACSCant.TM. II.
Results
[0635] In the negative control group (PBS administration group),
the proportion of PDGFR.alpha.-positive, CD44-positive cells in the
peripheral blood was 1.33% on average. Meanwhile, in the peptide
(1-44) administration group, the proportion was increased to 4.33%
on average (FIG. 15)
Discussion
[0636] When a peptide of amino acid positions 1 to 44 of HMGB1 was
synthesized and administered to mice, PDGFR.alpha.-positive,
CD44-positive cells were increased after 12 hours. In Example 8,
the peptide exhibited in vitro migration-promoting activity on
PDGFR.alpha.-positive, CD44-positive bone marrow mesenchymal stem
cells. The present Example demonstrates that the peptide also
recruits PDGFR.alpha.-positive, CD44-positive cells to the
peripheral blood in vivo. Both PDGFR.alpha. positivity and CD44
positivity are markers for bone marrow mesenchymal stem cells. Bone
marrow mesenchymal stem cells are known to be useful in
regenerative medicine. Thus, intravenous administration of the
peptide is expected to be effective to treat damaged tissues.
EXAMPLE 13
Creation of Middle Cerebral Artery Thread Occlusion Model
[0637] Eight- to ten-week-old male Wister rats were used. Rats were
anesthetized by isoflurane inhalation while warming on a keep-warm
mat with a body temperature monitor. After confirming that the
anesthetic effect was sufficient, cervical hair was removed to
expose the skin. The surgical site was sterilized with alcohol.
Along the cervical median line, the skin was incised with a
scalpel. After the right external carotid artery was ligated and
the right common carotid artery was pressurized to temporarily
block the blood flow, an occlusion thread made of #4 monofilament
nylon with siliconized tip was inserted into the right external
carotid artery toward the right internal carotid artery. While
releasing the pressure on the common carotid artery, the occlusion
thread was advanced along with the blood stream from the internal
carotid artery up to the bifurcation to the middle cerebral artery
so that the blood flow was blocked. Furthermore, the thread around
the right common carotid artery was ligated to completely block the
blood flow for 50 minutes. After removing the occlusion thread and
loosening the ligature at the common carotid artery, the skin was
sutured to complete the surgery.
Administration of Therapeutic Agents
[0638] 50 .mu.g of synthetic peptide (1-44) was administered at the
caudal vein. The first administration was carried out 6 hours after
the production of cerebral infarction. Then, the peptide was
administered five times at 24-hour intervals (five days in
total).
Determination of Size of Cerebral Infarction
[0639] After 14 days of the final drug administration, the rats
were given sufficiently deep anesthesia, and placed in a container
filled with carbon dioxide. The complete arrest of heartbeat and
breathing was confirmed. Brains were excised and immediately fixed
by immersion in buffered 10% formalin. After paraffin embedding,
thin sections were prepared and stained with hematoxylin-eosin.
Four sections were prepared from each brain at 1.92 mm anterior to
bregma (1.92), 0.60 mm anterior to bregma (0.60), 1.56 mm posterior
to bregma (-1.56), and 3.24 mm posterior to bregma (-3.24), and the
areas were compared.
Results
[0640] In the synthetic peptide (1-44) administration group (N=10),
only one mouse showed expansion of the infarction to the cortex,
while in the remaining 9 mice the infarction was limited in the
basal nucleus (FIG. 16 A1; (1.92 mm anterior to bregma), B1 (0.60
mm anterior to bregma), C1 (1.56 mm posterior to bregma), and D1
(3.24 mm posterior to bregma)). Meanwhile, in the negative control
group (N=11), 8 mice showed expansion of the infarction from the
basal nucleus up to the cortex (FIG. 16 A2; (1.92 mm anterior to
bregma), B2 (0.60 mm anterior to bregma), C2 (1.56 mm posterior to
bregma), and D2 (3.24 mm posterior to bregma)). Furthermore, on
each of the four prepared sections, the area of cerebral infarction
in the right brain was measured to determine the % ratio to normal
brain area in the right brain. In all sections, the infarction area
in the synthetic peptide administration group was significantly
reduced as compared to the negative control group (FIG. 17).
Discussion
[0641] It has recently been reported that the prognosis of patients
with cerebral infarction is improved by intravenous administration
of bone marrow mesenchymal stem cells of themselves. Thus, the
therapeutic effect of intramedullary cells on cerebral infarction
has been becoming clear. Further, while it is known from rodent
experiments that bone marrow mesenchymal stem cells differentiate
into osteoblasts, chondrocytes, adipocytes, and such, they have
also been revealed to differentiate into various cells such as
epithelial cells and neurons. Moreover, since bone marrow cells
secrete a variety of growth factors and cellular growth factors,
substances secreted by bone marrow cells that migrated to
infarction sites can be expected to produce neuroprotective
effects.
[0642] In the case of rats, it is known that by 48 hours after
ischemia, cerebral infarction is almost established in 80% to 90%
of its size, and then gradually expanded over subsequent 7 days.
Furthermore, it is known that there is an area called "core",
which, once ischemia occurs, inevitably becomes necrotized
irrespective of treatment, and an area called "penumbra", of which
necrosis could be avoided by treatment. Thus, preventing the
necrosis of penumbra before infarct expansion is an aim of cerebral
infarction therapy.
[0643] The cerebrum is primarily divided into basal nucleus and
cerebral cortex. In particular, the basal nucleus is more
vulnerable to hypoxia than the cerebral cortex, and is more easily
damaged by cerebral infarction. The result of the present Example
also demonstrates that the reduction of infarction by synthetic
peptide (1-44) was primarily observed in the cortex while the basal
nucleus necrotized in most cases. Since the cerebral cortex is the
center of sensation and movement, improvement of these functions is
very important for rehabilitation into society after treatment of
cerebral infarction. In light of the existing circumstances where
only a small number of effective therapeutic agents are available
for cerebral infarction, the need for the peptides of the present
invention as pharmaceutical agents is expected to be high.
[0644] In the experiment of the present Example, when the peptide
was administered 6 hours after production of cerebral infarction,
the effect of reducing the cerebral infarction size was seen after
19 days. The therapeutic effect is presumed to be due to the
neuroprotective action of bone marrow cells and tissue regeneration
caused by differentiation of the cells into neural tissues and
such. It was strongly suggested that peptides consisting of a
portion of HMGB1 could recruit cells to a damaged site not only
when administered at or near the damaged site but also when
administered into a vein which is a site different and distal to
the damaged site. Meanwhile, the peptides described in Examples 4,
5, and 6 include some peptides whose migration-promoting activity
seems too weak to be detected. It is considered that some of these
peptides have activity that is below the detection limit of the
assay method. The activity might be detected by optimizing the
medium to dissolve the peptides, the measurement time for the
migration-promoting activity, and the number of cells placed in the
upper layer of the chamber. Regarding tPA, which is a
pharmaceutical currently used for treating cerebral infarction,
there are strict administration criteria to ensure prevention of
side effects such as post-infarction hemorrhage: for example, it
has to be administered within four hours after the onset of
cerebral infarction, and diagnostic imaging is required. Since
cerebral infarction occurs suddenly, it is difficult to predict the
onset in advance. For this reason, most people who have developed
cerebral infarction often become no longer suitable for tPA when
they are seen in medical institutions because of expiry of the time
limit. On the other hand, in the present Example, the therapeutic
effect was obtained by administering the peptide 6 hours after
production of cerebral infarction. Since the present peptide is
considered to have no anticoagulant activity, it can be
administered even later than 6 hours. Thus, the peptide is expected
to be used for many people with cerebral infarction. Meanwhile, in
this Example, the peptide was administered to rats (about 250
g/head) at 50 .mu.g/head/administration. This corresponds to 200
.mu.g/kg weight, and is considered appropriate as a dose of
transvenous administration to patients with cerebral
infarction.
EXAMPLE 14
Construction of Expression Vectors for HMGB1 Fragments, Protein and
Peptide Expression, and Bone Marrow Mesenchymal Stem Cell Migration
Assay Method
[0645] The N terminal methionine (M) of human HMGB1 was deleted,
and MKHHHHHHENLYFQ (SEQ ID NO: 11) was added instead. HHHHHH (SEQ
ID NO: 12) is a tag (6.times. His tag) for use in purification of
an expressed protein or peptide using a nickel column. ENLYFQG (SEQ
ID NO: 13) is a sequence that is recognized by TEV protease (FIG.
18A). Furthermore, vectors were constructed in which a cDNA
encoding a protein or peptide of interest (2-215, 2-84, 2-44,
45-84, 2-62, 2-70, 2-81, 2-170, 93-215, or 85-169) were inserted
downstream of the T7 promoter and lac operator, the drug resistance
gene was a kanamycin resistance gene, and the replication origins
were pBR322 ori and fl ori. A human HMGB1 protein or peptide that
starts from the second amino acid can be prepared by cleaving with
TEV protease a protein or peptide obtained using the
above-described expression vector. BL-21(DE3) was transformed with
the constructed plasmids. The bacteria were cultured in LB
containing kanamycin while shaking at 37.degree. C. overnight, and
5 ml of bacterial suspension was transferred into 100 ml of LB. The
bacteria were cultured while shaking at 140 rpm and 37.degree. C.
The turbidity was measured with a turbidimeter, and, when OD
reached 0.5 to 0.7, isopropyl-.beta.-D-thiogalactopyranoside (IPTG)
was added at a final concentration of 1 mM. After five hours of
shaking culture at 37.degree. C. for 2-215, 2-84, 2-70, 2-81,
2-170, 93-215, and 85-169, or after overnight shaking culture at
15.degree. C. for 2-44, 45-84, and 2-62, the resulting bacteria
were collected. The expressed protein and peptides were assessed by
SDS-PAGE followed by protein staining and Western blotting with an
antibody against the tag or anti-HMGB1 antibody.
Purification of Respective HMGB1 Fragments (2-215, 2-84, 2-44,
45-84, 2-62, 2-70, 2-81, 2-170, and 93-215)
[0646] 3 ml of equilibration buffer (PBS (137 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.4, 2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4), 10 mM
imidazole; pH 7.4) was added to collected bacterial cells. The
bacterial cells were sonicated, and centrifuged at 15,000 rpm and
4.degree. C. for 10 minutes. The supernatant was collected. 1 ml of
His-Pur.TM. Ni-NTA Resin (Thermo Scientific) was loaded in Micro
Bio-Spin column (Bio-Rad) and equilibrated with the equilibration
buffer. A protein solution was loaded onto the column. After two
minutes of centrifugation at 2000 rpm, the resin was washed with
washing buffer (PBS, 25 mM imidazole; pH 7.4). The column was
eluted with elution buffer (PBS, 250 mM or 500 mM imidazole) in a
stepwise manner. Each fraction was subjected to SDS-PAGE (15%
e-PAGEL.RTM. (ATTO)) to confirm the eluted protein. After affinity
purification with a nickel column, ion-exchange chromatography was
performed using Q sepharose.TM. Fast Flow (GE Healthcare) for
2-215; using Q sepharose.TM. Fast Flow (GE Healthcare) and SP
sepharose.TM. Fast Flow (GE Healthcare) for 93-215; or using SP
sepharose.TM. Fast Flow (GE Healthcare) for the remaining
peptides.
Human HMGB1 Fragments (2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81,
and 2-170)
[0647] 1 ml of each sepharose was loaded in Micro Bio-Spin column
(Bio-Rad) and was equilibrated with PBS. After loading a solution
of affinity purified protein, the column was washed with PBS, and
eluted with elution buffer (20 mM HEPES, 1 M NaCl; pH 7.5).
Each fraction was examined by SDS-PAGE.
Human HMGB1 Fragment (93-215)
[0648] A solution of affinity purified protein was subjected to
anion and cation exchange using Q and SP sepharose, respectively.
Then, the flowthrough fraction from SP sepharose.TM. Fast Flow was
loaded onto Q sepharose.TM. Fast Flow to perform anion
exchange.
Each fraction was examined by SDS-PAGE.
Human HMGB1 Fragment (85-169)
[0649] One ml of buffer (PBS, 10 mM imidazole; pH 7.4) was added to
0.1 g each of the collected bacterial cells. The bacterial cells
were sonicated and centrifuged at 20,000 rpm and 4.degree. C. for
one hour. The supernatant was collected and purified by column
chromatography using BioLogic DuoFlow (Bio-Rad). First, affinity
purification was carried out with 5 ml of HisTrap.TM. FF (GE
Healthcare) using a bacterial lysis buffer (PBS, 10 mM imidazole
(pH 7.4)) as Buffer A and PBS (pH 7.4) containing 500 mM imidazole
as Buffer B. After the column was equilibrated with Buffer A, a
protein solution was loaded onto it. Washing and purification were
performed with the program described below. The program used is as
follows: [0650] Isocratic Flow (Buffer A: 97%, Buffer B: 3%, 20
ml).fwdarw.Linear Gradient (Buffer A: 97%.fwdarw.0%, Buffer B:
3%.fwdarw.100%, 20 ml).fwdarw.Isocratic Flow (Buffer B: 100%, 20
ml).fwdarw.Fraction Collection (2 ml each; 20 to 40 ml) Each
fraction was examined by SDS-PAGE.
[0651] Then, ion-exchange purification was performed using 5 ml
column of HiTrap.TM. SP HP (GE Healthcare) for 85-169, and PBS (pH
7.4) as Buffer A, and 20 mM HEPES buffer (pH 7.5) containing 1 M
NaCl as Buffer B. After the column was equilibrated with an
appropriate amount of Buffer A, a solution of affinity purified
protein was loaded onto it. Washing and purification were performed
with the program described below. The program used is as follows:
[0652] Isocratic Flow (Buffer A: 100%, Buffer B: 0%, 10
ml).fwdarw.Isocratic Flow (Buffer A: 50%, Buffer B: 50%, 2
ml).fwdarw.Isocratic Flow (Buffer A: 0%, Buffer B: 100%, 20
ml).fwdarw.Fraction Collection (1 ml each; 10 to 32 ml) Each
fraction was examined by SDS-PAGE.
Concentration Determination
[0653] The concentration of each fragment was determined in BSA
equivalents using Bradford method (Bio-Rad Protein Assay).
Migration Assay
[0654] Each of the above-described peptides was assessed for
migration-promoting activity on bone marrow mesenchymal stem cell
line MSC-l. Each fragment in phosphate buffer containing 500 mM
NaCl was diluted at a final concentration of 2 .mu.M with two
volumes of DMEM, and added to the lower layer of a chamber, while
MSC-1 suspended in DMEM containing 10% FBS was placed in the upper
layer. Polycarbonate membrane with 8-.mu.m pores was inserted
between both layers. After four hours of incubation in an incubator
under 5% CO.sub.2 at 37.degree. C., cells that migrated from the
upper layer to the lower layer were detected by using Diff-Quik
stain.TM..
Results
[0655] All of 2-215, 2-84, 2-44, 45-84, 2-62, 2-70, 2-81, 2-170,
and 93-215 showed migration-promoting activity on bone marrow
mesenchymal stern cells (FIG. 18B). When the migration-promoting
activity of 2-215 is taken as 1, the activities of 2-84, 2-44, and
45-84 per molar concentration were 2.37, 1.82, and 2.04 times,
respectively, and those per equal mass were 5.5, 7.1, and 8.1
times, respectively (FIGS. 18C and 18D).
Discussion
[0656] Fragmentation of the N-terminal of human HMGB1 (2-215)
produced in E. coli resulted in an increase in the
migration-promoting activity. Furthermore, at least two regions on
the N terminal side, 2-44 and 45-84, exhibited migration-promoting
activity. This is consistent with the result for the HMGB1
fragments produced by eukaryotic cell culture (HEK293 cells). It is
presumed that the fragmentation results in exposure of epitopes to
the receptor on MSC-1, and thereby facilitates the receptor
binding. Although some proteins lose their activity by
fragmentation, the present protein showed rather increased activity
as a result of fragmentation. It is known that proteins expressed
in eukaryotic cells such as HEK293 undergo post-translational
modification such as glycosylation. The presence of such
modification may affect the activity of receptor ligands. Thus, the
fact that not only the protein produced in E. coli, which does not
perform the same post-translational modification as eukaryotic
cells, but also the fragments produced in E. coli retain the
activity suggests that post-translational modification is not
essential for the activity of the fragments. The findings described
above demonstrate that fragmentation of HMGB1 enables development
of highly active agents for recruiting bone marrow mesenchymal stem
cells. Furthermore, since post-translational modification is not
essential, production methods using E. coli or chemical synthesis
are possible, enabling production of preparations more stable in
quality at lower costs. Moreover, comparison between the peptides
described in this Example and those described in other Examples
(for example, comparison between 1-44 and 2-44 or between 1-84 and
2-84) revealed that the migration-promoting activity was not
affected by the presence of the first methionine in the HMGB1
protein. Hence, when a peptide has migration-promoting activity, a
peptide in which the first methionine is removed from the peptide
is also considered to have migration-promoting activity.
Alternatively, when a peptide lacking the first methionine has
migration-promoting activity, a peptide to which the first
methionine is added is also considered to have migration-promoting
activity.
EXAMPLE 15
Methods
[0657] The N terminal methionine (M) was deleted from human HMGB1,
and instead MKHHHHHHENLYFQ (SEQ ID NO: 11) was added to the N
terminus. HHHHHH (SEQ ID NO: 12) is a tag (6.times. His tag) for
use in purification of an expressed protein or peptide using a
nickel column. ENLYFQG (SEQ 1D NO: 13) is a sequence that is
recognized by TEV protease (FIG. 18A). Furthermore, vectors were
constructed in which a cDNA encoding a protein or peptide of
interest (89-215, 89-205, 89-195, or 89-185) was inserted
downstream of T7 promoter and lac operator, and the drug resistance
gene was a kanamycin resistance gene, and the replication origins
were pBR322 ori and fl ori. A human HMGB1 protein or peptide that
starts from the second amino acid can be prepared by cleaving with
TEV protease a protein or peptide obtained using the
above-described expression vector.
[0658] BL-21(DE3) was transformed with the constructed plasmids.
The bacteria were cultured in LB containing kanamycin while shaking
at 37.degree. C. overnight, and 5 ml of bacterial suspensions were
transferred into 100 ml of LB. The bacteria were cultured while
shaking at 140 rpm and 37.degree. C. The turbidity was measured
with a turbidimeter, and, when OD reached 0.5 to 0.7,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added at a
final concentration of 1 mM. After overnight shaking culture at
15.degree. C., the bacterial cells for human HMGB1 fragments
(89-215, 89-205, 89-195, and 89-185) were harvested. The expressed
protein and peptides were examined by SDS-PAGE followed by protein
staining and Western blotting with an antibody against the tag or
anti-HMGB1 antibody.
Purification of Respective HMGB1 Fragments (89-215, 89-205, 89-195,
and 89-185)
[0659] Two ml of buffer (PBS, 10 mM imidazole; pH 7.4) was added to
0.1 g each of the collected bacterial cells. The bacterial cells
were sonicated and centrifuged at 20,000 rpm and 4.degree. C. for
one hour. The supernatant was collected and purified by column
chromatography using BioLogic DuoFlow (Bio-Rad).
Human HMGB1 Fragment (89-215)
[0660] One ml of buffer (PBS, 10 mM imidazole; pH 7.4) was added to
0.1 g each of the collected bacterial cells. The bacterial cells
were sonicated and centrifuged at 20,000 rpm and 4.degree. C. for
one hour. The supernatant was collected and purified by column
chromatography using BioLogic DuoFlow (Bio-Rad). First, affinity
purification was carried out with 5 ml of HisTrap.TM. FF (GE
Healthcare) using a bacterial lysis buffer (PBS, 10 mM imidazole
(pH7.4)) as Buffer A and PBS (pH 7.4) containing 500 mM imidazole
as Buffer B. After the column was equilibrated with Buffer A, a
protein solution was loaded onto it. Washing and purification were
performed with the program described below. The program used is as
follows: [0661] Isocratic Flow (Buffer A: 97%, Buffer B: 3%, 20
ml).fwdarw.Linear Gradient (Buffer A: 97%.fwdarw.0%, Buffer B:
3%.fwdarw.100%, 20 ml).fwdarw.Isocratic Flow (Buffer B: 100%, 20
ml).fwdarw.Fraction Collection (2 ml each; 20 to 40 ml) Each
fraction was examined by SDS-PAGE.
[0662] Then, ion-exchange purification was performed using 5 ml of
HiTrap.TM. Q HP (GE Healthcare) for 89-215, and PBS (pH 7.4) as
Buffer A, and 20 mM HEPES buffer (pH 7.5) containing 1 M NaCl as
Buffer B. After the column was equilibrated with an appropriate
amount of Buffer A, a solution of affinity purified protein was
loaded onto it. Washing and purification were performed with the
program described below. The program used is as follows: [0663]
Isocratic Flow (Buffer A: 100%, Buffer B: 0%, 10
ml).fwdarw.Isocratic Flow (Buffer A: 50%, Buffer B: 50%, 2
ml).fwdarw.Isocratic Flow (Buffer A: 0%, Buffer B: 100%, 20
ml).fwdarw.Fraction Collection (1 ml each; 10 to 32 ml) Each
fraction was examined by SDS-PAGE.
Human HMGB1 Fragments (89-205, 89-195, and 89-185)
[0664] Solutions of soluble proteins were prepared in the same
manner as human HMGB1 fragment (89-215). Then, the following
gradient elution was performed by each column chromatography.
[0665] First, affinity purification was carried out using 5 ml of
HisTrap.TM. FF, Buffer A (PBS (pH7.4) containing 10 mM imidazole),
and Buffer B (PBS (pH 7.4) containing 500 mM imidazole). After the
column was equilibrated with Buffer A, a protein solution was
loaded onto it. Washing and purification were performed with the
program described below. The program used is as follows: [0666]
.fwdarw.Isocratic Flow (Buffer A: 97%, Buffer B: 3%, 50
ml).fwdarw.Linear Gradient (Buffer A: 97%.fwdarw.0%, Buffer B:
3%.fwdarw.100%, 120 ml).fwdarw.Fraction Collection (5 ml each; 50
to 170 ml) Each fraction was examined by SDS-PAGE.
[0667] Then, ion-exchange purification was performed using 5 ml
column of HiTrap.TM. Q HP for human HMGB1 fragments (89-215) and
(89-205), and 5 ml column of HiTrap.TM. SP HP for the other
fragments. PBS (pH 7.4) was used as Buffer A, while 7.times. PBS
(pH 7.4) was used as Buffer B. After the column was equilibrated
with an appropriate amount of Buffer A, a solution of affinity
purified protein was loaded onto it. Washing and purification were
performed with the program described below. The program used is as
follows: [0668] Isocratic Flow (Buffer A: 100%, Buffer B: 0%, 50
ml).fwdarw.Linear Gradient (Buffer A: 100%.fwdarw.0%, Buffer B:
0%.fwdarw.100%, 50 ml).fwdarw.Isocratic Flow (Buffer A: 0%, Buffer
B: 100%, 5 ml).fwdarw.Fraction Collection (3 ml each; 50 to 105 ml)
Each fraction was examined by SDS-PAGE.
Concentration Determination
[0669] The concentration of each fragment was determined in BSA
equivalents using Bradford method (Bio-Rad Protein Assay).
Migration Assay
[0670] Each of the above-described peptides was examined for
migration-promoting activity on bone marrow mesenchymal stem cell
line MSC-1. Each fragment in phosphate buffer containing 500 mM
NaCl was diluted at a final concentration of 2 .mu.M with two
volumes of DMEM, and added to the lower layer of a chamber, while
MSC-1 dispersed in DMEM containing 10% FBS was placed in the upper
layer. Polycarbonate membrane with 8-.mu.m pores was inserted
between the upper and lower layers. After four hours of incubation
in an incubator under 5% CO.sub.2 at 37.degree. C., cells that
migrated from the upper layer to lower layer were detected by using
Diff-Quik stain.TM..
Results
[0671] After affinity purification of human HMGB1 fragment (89-215)
with a nickel column, gradient elution with increasing salt
concentration was performed using ion-exchange chromatography (Q
column). A 15.5-kDa peptide was fractionated into fractions 6 and
7; 15.5-, 16-, and 17-kDa peptides were fractionated into fractions
8 and 9; and 16- and 17-kDa peptides were fractionated into
fractions 10 and 11 (FIG. 19A).
[0672] A mixed sample (6+7) of fractions 6 and 7, and fractions 9
and 10 were tested for migration-promoting activity on bone marrow
mesenchymal stem cells (MSC-1). The activity of the mixed sample of
fractions 6 and 7 (6+7) was strong, while those of fractions 9 and
10 were weak (FIG. 19B)
[0673] After affinity purification of human HMGB1 fragment (89-205)
with a nickel column, gradient elution with increasing salt
concentration was carried out using ion-exchange chromatography (Q
column). The shortest fragment (*4) was eluted first in fraction 6,
and the next shortest fragment (*5) and then the longest fragment
(*6) were eluted in fraction 7. Meanwhile, 89-195 and 89-185 were
purified as a single fragment by affinity purification using a
nickel column (FIG. 19C).
[0674] Fractions 6, 7, and 8 were examined for migration-promoting
activity on bone marrow mesenchymal stem cells (MSC-1). Fraction 6
showed strong activity, and the activity was decreased as the
fraction number increased. Meanwhile, 89-195 and 89-185 showed
stronger activity than any fragments between positions 89 and 215
(FIG. 19D)
Discussion
[0675] Human HMGB1 fragments (89-215, 89-205, 89-195, and 89-185)
were prepared using E. coli. 89-215 and 89-205 exhibited weak
migration-promoting activity on bone marrow mesenchymal stem cells.
However, they underwent cleavage that seemed to be caused by
protease derived from E. coli (FIGS. 19A and 19C), and short
fragments exhibited strong activity (FIGS. 19B and 19D). Meanwhile,
89-195 and 89-185 showed strong migration-promoting activity on
bone marrow mesenchymal stem cells (FIGS. 19C and 19D). The
C-terminal amino acids of positions 186 to 215 of HMGB1 contain a
repeat sequence of glutamine and aspartic acid. The sequence is
said to contribute to protein stabilization. The present study for
the first time demonstrated that this portion suppressed the
migration-promoting activity of HMGB1 fragment (89-215) and thus
the activity could be increased by removing this sequence. The
C-terminal glutamic acid/aspartic acid repeat sequence (the amino
acid sequence from positions 186 to 215) in HMGB1 is called "acidic
tail", and has been reported to be essential for the binding to
RAGE. Meanwhile, based on the fact that RAGE serves as the receptor
responsible for HMGB1-mediated migration of dendritic cells and
such, it was once predicted that the C-terminal portion and the
RAGE ligand portion would be indispensable to exert the
migration-promoting activity. Surprisingly, in fact, the lack of
the C-terminal was found to be more advantageous for the
migration-promoting activity on bone marrow mesenchymal stem cells.
This was not known until it was discovered that, when the
C-terminal-containing HMGB1 fragment was produced in E. coli,
degradation products that seemed to be produced by E. coli-derived
protease exhibited stronger migration-promoting activity than the
intact HMGB1 fragment and that when an HMGB1 fragment lacking the C
terminus was produced, it exhibited stronger activity than the
C-terminal-intact HMGB1 fragment. In general, a particular activity
of protein is contributed by only a single region; however,
surprisingly, HMGB1 had multiple regions that contribute to its
migration-promoting activity on bone marrow mesenchymal stem cells,
and more surprisingly, the activity of each region per equal number
of molecules was about twice that of the full-length HMGB1. In
addition, although biologically active peptides in general become
more unstable and less active as their length becomes shorter,
surprisingly, some shorter fragments had stronger activity than
longer fragments.
EXAMPLE 16
Methods
[0676] The N terminal methionine (M) was deleted from human HMGB1,
and instead MKHHHHHHENLYFQ (SEQ ID NO: 11) was added to the N
terminus. HHHHHH (SEQ ID NO: 12) is a tag (6.times. His tag) for
use in purification of an expressed protein or peptide using a
nickel column. ENLYFQG (SEQ ID NO: 13) is a sequence that is
recognized by TEV protease (FIG. 18A). Furthermore, vectors were
constructed in which a cDNA encoding a protein or peptide of
interest (85-169, 2-215) was inserted downstream of T7 promoter and
lac operator, the drug resistance gene was a kanamycin resistance
gene, and the replication origins were pBR322 ori and fl ori. A
human HMGB1 protein or peptide that starts from the second amino
acid can be prepared by cleaving with TEV protease a protein or
peptide obtained using the above-described expression vector.
[0677] BL-21(DE3) was transformed with the constructed plasmids.
The bacteria were cultured in LB containing kanamycin while shaking
at 37.degree. C. overnight, and 5 ml of bacterial suspensions were
transferred into 100 ml of LB. The bacteria were cultured while
shaking at 140 rpm and 37.degree. C. The turbidity was measured
with a turbidimeter, and, when OD reached 0.5 to 0.7,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added at a
final concentration of 1 mM. After overnight shaking culture at
15.degree. C., the bacterial cells for human HMGB1 fragment
(85-169) were harvested. The expressed protein and peptides were
examined by SDS-PAGE followed by protein staining and Western
blotting with an antibody against the tag or anti-HMGB1
antibody.
[0678] One ml of buffer (PBS, 10 mM imidazole; pH 7.4) was added to
0.1 g each of the collected bacterial cells. The bacterial cells
were sonicated and centrifuged at 20,000 rpm and 4.degree. C. for
one hour.
[0679] The supernatant was collected and purified by column
chromatography using BioLogic DuoFlow (Bio-Rad). First, affinity
purification was carried out with 5 ml of HisTrap.TM. FF (GE
Healthcare) using a bacterial lysis buffer (PBS, 10 mM imidazole
(pH7.4)) as Buffer A and PBS (pH 7.4) containing 500 mM imidazole
as Buffer B. After the column was equilibrated with Buffer A, a
protein solution was loaded onto it. Washing and purification were
performed with the program described below. The program used is as
follows: [0680] Isocratic Flow (Buffer A: 97%, Buffer B: 3%, 20
ml).fwdarw.Linear Gradient (Buffer A: 97%.fwdarw.0%, Buffer B:
3%.fwdarw.100%, 20 ml).fwdarw.Isocratic Flow (Buffer B: 100%, 20
ml).fwdarw.Fraction Collection (2 ml each; 20 to 40 ml) Each
fraction was examined by SDS-PAGE.
Concentration Determination
[0681] The concentration of each recombinant protein was determined
in BSA equivalents using Bradford method (Bio-Rad Protein
Assay).
Migration Assay
[0682] Each of the above-described peptides was examined for
migration-promoting activity on bone marrow mesenchymal stem cell
line MSC-1. Each fragment in phosphate buffer containing 500 mM
NaCl was diluted at a final concentration of 2 .mu.M with two
volumes of DMEM, and added to the lower layer of a chamber, while
MSC-1 dispersed in DMEM containing 10% FBS was placed in the upper
layer. Polycarbonate membrane with 8-.mu.m pores was inserted
between the upper and lower layers. After four hours of incubation
in an incubator under 5% CO.sub.2 at 37.degree. C., cells that
migrated from the upper to lower layer were detected by using
Diff-Quik stain.TM..
Results
[0683] Human HMGB1 fragment (85-169) exhibited stronger
migration-promoting activity on bone marrow mesenchymal stem cells
than human HMGB1 fragment (2-215) (FIG. 20A). When the
migration-promoting activity is taken as 1, the activity per molar
was 1.59 times, and the activity per equal mass was 3.6 times
(FIGS. 20B and 20C).
Discussion
[0684] Like human HMGB1 fragments (89-195) and (89-185), human
HMGB1 fragment (85-169) showed stronger migration-promoting
activity on bone marrow mesenchymal stem cells than (2-215). From
the above-described finding, it is presumed that at least one
sequence with the activity of recruiting bone marrow mesenchymal
stem cells is present within the amino acids of positions 85-185.
In Example 1, HMGB1 fragment 85-169 produced in HEK293 did not show
migration-promoting activity. In the present Example, however,
HMGB1 fragment 85-169 produced in E. coli exhibited
migration-promoting activity. This difference is presumably due to
significant diminishment or loss of the migration-promoting
activity depending on the production method. Since eukaryotes such
as HEK293 and prokaryotes such as E. coli are different in
post-translational modification, folding, and such, even the same
proteins or peptides produced by them often have different
properties.
[0685] The present study revealed that the amino acid sequence of
HMGB1 has at least three sequences with the activity of recruiting
bone marrow mesenchymal stem cells, and their activity was
regulated through the suppression by the C-terminal glutamic
acid/aspartic acid repeat sequence. By preparing HMGB1 fragments
that lack the C-terminal suppressive sequence, it is possible to
produce highly active preparations having the effect of recruiting
bone-marrow stem cells.
EXAMPLE 17
Mouse Mesenchymal Stem Cell Migration Activity (1)
Methods
Purification of HMGB1 Fragments
[0686] Inverse PCR was carried out using KOD-Plus-ver.2 (Toyobo).
The above-described expression vector for HMGB1 fragment containing
the amino acids of positions 2 to 215 of human HMGB1 was used as a
template plasmid. cDNAs encoding the amino acids of positions 2 to
205, the amino acids of positions 2 to 195, the amino acids of
positions 2 to 185 were amplified by PCR, together with the
N-terminal histidine tag, TEV protease recognition sequence, and
the plasmid backbone. The gene products prepared from the PCR
products are proteins in which a histidine tag, a TEV protease
recognition sequence, and a human HMGB1 fragment are aligned in
tandem. The template plasmid was digested by adding restriction
enzyme DpnI (Toyobo) to the PCR products. Then, the PCR products
were phosphorylated using T4 Polynucleotide kinase (NEB), and
self-ligated with ligase (2.times. Quick Ligase (NEB); or Ligation
Convenience kit (Nippongene)). The products were used to transform
E. coli JM 109, and colonies were obtained via kanamycin selection.
Plasmid extraction was carried out using GenElute Plasmid Miniprep
kit (SIGMA-ALDRICH). After determining the nucleotide sequences by
sequencing analysis, E. coli BL21 (DE3) was transformed with the
plasmids to give colonies.
Induction of Expression
[0687] Each colony was cultured in a medium containing kanamycin at
a final concentration of 50 mg/l while shaking at 37.degree. C.
overnight. 5 ml of the bacterial suspension was transferred into
100 ml of LB. The bacteria were cultured at 37.degree. C. while
shaking at 140 rpm. The turbidity was measured with a turbidimeter,
and, when OD reached 0.5 to 0.7,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added at a
final concentration of 1 mM. After overnight shaking culture at
15.degree. C., the bacterial cells were collected.
Purification of Recombinant Proteins
[0688] 12 ml (25-50 mg/ml) of equilibration buffer (PBS (137 mM
NaCl, 8.1 mM Na.sub.2HPO.sub.4, 2.68 mM KCl, 1.47 mM
KH.sub.2PO.sub.4), 10 mM imidazole; pH7.4) was added to the
collected bacterial cells, and leupeptin hydrochloride was added at
a final concentration of 5 .mu.g/ml thereto. The bacterial cells
were sonicated and centrifuged at 15,000 rpm and 4.degree. C. for
60 minutes. The supernatant was collected and an aliquot thereof
was examined by Western blotting using an anti-human HMGB1 antibody
to confirm the expression of the protein of interest. The remaining
supernatant was sterilized by filtering through a 0.45-.mu.m
filter. The protein of interest was purified by column
chromatography using BioLogic DuoFlow (Bio-Rad).
[0689] First, affinity purification was performed with 5 ml of
HisTrap.TM. FF using Buffer A (PBS, 10 mM imidazole (pH7.4)) and
Buffer B (PBS, 500 mM imidazole (pH 7.4)). After the column was
equilibrated with Buffer A, a protein solution was loaded onto it.
Washing and purification were performed with the program described
below.
Program:
[0690] Isocratic Flow (Buffer A: 97%, Buffer B: 3%, 50 ml)
[0691] Linear Gradient (Buffer A: 97%.fwdarw.0%, Buffer B:
3%.fwdarw.100%, 120 ml)
[0692] Fraction Collection (5 ml each; 50 to 170 ml)
Each fraction was examined by SDS-PAGE (5-20% e-PAGE.RTM.
(ATTO)).
[0693] Then, ion-exchange purification was performed using 5 ml
column of HiTrap.TM. Q HP for GNX-E-022 only, and 5 ml of
HiTrap.TM. SP HP for the others. PBS (pH 7.4) was used as Buffer A,
while 7.times. PBS (pH 7.4) was used as Buffer B. After the column
was equilibrated with an appropriate amount of Buffer A, a solution
of affinity purified protein was loaded onto it. Washing and
purification were performed with the program described below.
Program:
[0694] Isocratic Flow (Buffer A: 100%, Buffer B: 0%, 50 ml, 4
ml/min)
[0695] Linear Gradient (Buffer A: 100%.fwdarw.0%, Buffer B:
0%.fwdarw.100%, 50 ml, 4 ml/min)
[0696] Isocratic Flow (Buffer A: 0%, Buffer B: 100%, 5 ml, 4
ml/min)
[0697] Fraction Collection (3 ml each; 50 to 105 ml)
Each fraction was subjected to SDS-PAGE followed by protein
staining to confirm purified proteins.
Concentration Determination
[0698] The concentration of recombinant proteins was determined in
BSA equivalents using Bradford method (Bio-Rad Protein Assay).
Migration Assay
[0699] Each of the above-described peptides was examined for
migration-promoting activity on bone marrow mesenchymal stem cell
line MSC-1. Each fragment in phosphate buffer containing 500 mM
NaCl was diluted at a final concentration of 2 .mu.M with two
volumes of DMEM, and added to the lower layer of a chamber, while
MSC-1 dispersed in DMEM containing 10% FBS was placed in the upper
layer. Polycarbonate membrane with 8-.mu.m pores was inserted
between the upper and lower layers. After four hours of incubation
in an incubator under 5% CO.sub.2 at 37.degree. C., cells that
migrated from the upper to lower layer were detected by using
Diff-Quik stain.TM..
Results
[0700] Fragments 2-195 and 2-185 showed stronger
migration-promoting activity than fragments 2-215 and 2-205 (FIG.
21A).
Discussion
[0701] The fragment from positions 186 to 215 is an aspartic
acid/glutamic acid repeat sequence of 30 amino acids in total, and
is called "acidic tail". The data described above suggests that the
migration-promoting activity of HMGB1 on bone marrow mesenchymal
stem cells is strongly suppressed by the acidic tail, and in
particular the C-terminal 20-amino acid sequence is involved in the
suppression. Based on the data described earlier, several active
domains for the migration-promoting effect of HMGB1 on bone marrow
mesenchymal stem cells were identified. Further detailed
experiments were needed to clarify whether the acidic tail
suppresses the activity of all these domains. Human mesenchymal
stem cell migration activity (2)
Methods
[0702] Human HMGB1 fragments (2-215, 2-84, 2-44, 45-84, 85-169,
89-185, 89-195, and 89-205) were produced in E. coli and purified
using appropriate columns in the same manner as described in
Examples 14, 15, and 16 above. However, 89-215 was purified by the
same method as used for 89-205 in Example 15.
Concentration Determination
[0703] The concentration of each fragment was determined in BSA
equivalents using Bradford method (Bio-Rad Protein Assay).
Migration Assay
[0704] Using human-derived bone marrow mesenchymal stem cells, each
fragment was examined by the same migration assay performed on
mouse-derived bone marrow mesenchymal stem cells (MSC-1) as
described above. The fragments were used at a final concentration
of 2 .mu.M. The human-derived bone marrow mesenchymal stem cells
used were hMSC (human Mesenchymal Stem Cell, Takara) at fourth
passage. The growth medium used was a mesenchymal stem cell growth
medium (MF medium, TOYOBO). The cells were cultured in an incubator
under 5% CO.sub.2 at 37.degree. C. The medium was changed with a
fresh medium every two to four days. The cells were passaged when
they reached 80% confluency.
Results
[0705] Regarding the human HMGB1 fragments (2-215, 2-84, 2-44, and
45-84), the activity of HMGB1 fragments (2-84, 2-44, and 45-84) was
greater than human HMGB1 fragment (2-215) as with the case of
Example 14. As to human HMGB1 fragments (89-185, 89-195, 89-205,
and 89-215), active human HMGB1 fragments (89-185 and 89-195) with
a shortened C-terminal acidic tail exhibited stronger activity as
with the case of Example 15. Meanwhile, the activity of human HMGB1
fragment (85-169) was greater than that of human HMGB1 fragment
(2-215) as with the case of Example 16 (FIG. 21B).
Discussion
[0706] All fragments showed migration-promoting activity on
human-derived bone marrow mesenchymal stem cells as with the case
of mouse-derived bone marrow mesenchymal stem cells. It was
demonstrated that independent domains having migration-promoting
activity on human bone marrow mesenchymal stem cells were located
at least in human HMGB1 fragments (2-44, 45-84, and 85-169). Since
typical proteins have only one site for particular activity, the
existence of multiple active sites is surprising. Moreover, it is
also surprising that the activity of each fragment is greater than
that of the sequence (2-215), which consists of nearly the full
length. Meanwhile, although the RAGE-binding domain is the amino
acid sequence from positions 150 to 183, the amino acid sequence of
positions 89 to 169 also has migration-promoting activity on human
bone marrow mesenchymal stem cells, suggesting that the
migration-promoting activity may not require RAGE. Regarding human
HMGB1 fragments (89-185, 89-195, 89-205, and 89-215), the fragments
lacking the C-terminal acidic tail showed stronger activity, as
with the case of mouse-derived cells. This finding suggests that
the C terminus also suppresses the migration-promoting activity on
human bone marrow mesenchymal stem cells, and therefore more active
HMGB1 fragments may be produced by shortening or eliminating the
C-terminal acidic tail.
Altered migration-promoting activity of fusion fragments in which
human HMGB1 acidic tail fragment 186-215, 186-205, or 186-195 is
added to human HMGB1 fragment 2-84
Methods
[0707] Fusion cDNAs were constructed such that human HMGB1 fragment
186-215, 186-205, or 186-195 was added to the C terminus of human
HMGB1 fragment 2-84. As described above, expression vectors were
designed such that, in a fragment expressed in E. coli, the N
terminal methionine (M) of human HMGB1 was deleted and instead
MKHHHIIHHENLYFQ (SEQ ID NO: 11) was added to its N terminus. HHHHHH
(SEQ ID NO: 12) is a tag (6.times. His tag) for use in purification
of an expressed protein or peptide using a nickel column. ENLYFQG
(SEQ ID NO: 13) is a sequence that is recognized by TEV protease
(FIG. 18A). Furthermore, vectors were constructed in which the cDNA
described above was inserted downstream of T7 promoter and lac
operator, the drug resistance gene was a kanamycin resistance gene,
and the replication origins were pBR322 ori and fl ori. A human
HMGB1 protein or peptide that starts from the second amino acid can
be prepared by cleaving with TEV protease a protein or peptide
obtained using the above-described expression vector.
[0708] BL-21(DE3) was transformed with the constructed plasmids.
The bacteria were cultured in LB containing kanamycin while shaking
at 37.degree. C. overnight, and 5 ml of bacterial suspensions were
transferred into 100 ml of LB. The bacteria were cultured while
shaking at 140 rpm and 37.degree. C. The turbidity was measured
with a turbidimeter, and, when OD reached 0.5 to 0.7,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was added at a
final concentration of 1 mM. After overnight shaking culture at
15.degree. C., the bacterial cells were harvested.
Purification of Respective HMGB1 Fragments (2-84+186-215,
2-84+186-205, and 2-84+186-195)
[0709] Equilibration buffer (PBS (137 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.4, 2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4), 10 mM
imidazole; pH7.4) was added to the collected bacterial cells so
that the final concentration was 5 .mu.g/ml. The bacterial cells
were sonicated and centrifuged at 15,000 rpm and 4.degree. C. for
60 minutes. The supernatant was collected, and the remaining
supernatant was sterilized by filtering through a 0.45-.mu.m
filter. The protein of interest was purified by column
chromatography using BioLogic DuoFlow (Bio-Rad).
[0710] Then, ion-exchange purification was performed using 5 ml of
HiTrap.TM. Q HP (GE Healthcare) for 2-84+186-215, 2-84+186-205, and
2-84+186-195. PBS (pH 7.4) was used as Buffer A, while 7.times. PBS
(pH 7.4) was used as Buffer B. After the column was equilibrated
with an appropriate amount of Buffer A, a solution of affinity
purified protein was loaded onto it. Washing and purification were
performed with the program described below. The program used is as
follows: [0711] Isocratic Flow (Buffer A: 100%, Buffer B: 0%, 50
ml, 4 ml/min)
[0712] Linear Gradient (Buffer A: 100%.fwdarw.0%, Buffer B:
0%.fwdarw.100%, 50 ml, 4 ml/min)
[0713] Isocratic Flow (Buffer A: 0%, Buffer B: 100%, 5 ml, 4
ml/min)
[0714] Fraction Collection (3 ml each; 50 to 105 ml)
Each fraction was examined by SDS-PAGE followed by protein staining
of the gel to confirm purified proteins.
[0715] Each fragment was subjected to Western blotting using an
antibody that recognizes human HMGB1 to confirm whether it is the
fragment of interest.
Migration assay of HMGB1 fragment (2-84), and fusion HMGB1
fragments (2-84+186-215, 2-84+186-205, and 2-84+186-195) using
MSC-1
[0716] Fragments prepared by the methods described above were used
to perform migration assay using bone marrow mesenchymal stem cell
line MSC-1. The migration assay was performed in the same manner as
described above.
Results
[0717] Fragment 2-84 exhibited migration-promoting activity, while
none of the fusion human HMGB1 fragments with 10 amino acids, 20
amino acids, or 30 amino acids of the acidic tail sequence showed
the activity (FIG. 21C).
Discussion
[0718] As shown in the above-mentioned Examples, human HMGB1
fragment 89-215 has only extremely weak migration-promoting
activity on mesenchymal stem cells; however, successive truncation
of the C-terminal acidic tail increases the migration-promoting
activity. The findings described above and the present Example
demonstrate that the acidic tail has the function to reduce the
migration-promoting activity of fragments 2-84 and 89-185. Fragment
2-84 has multiple core regions with migration-promoting activity.
Since fusion of fragment 2-84 with fragment 186-215 resulted in
almost complete loss of the migration-promoting activity, it is
presumed that the suppression acts on all the core regions in 2-84.
It is a surprising discovery that a single molecule of HMGB1
contains at least three or more core sequences with
migration-promoting activity on bone marrow mesenchymal stem cells.
Another very surprising discovery is that the C-terminal acidic
tail of only 30 amino acids almost completely suppresses the
migration-promoting activity of at least two core sequences in the
N-terminal fragment 2-84 of HMGB1 and suppresses the
migration-promoting activity of the core sequence in fragment
85-185 as well, resulting in a decrease in the overall
migration-promoting activity of HMGB1. HMGB1 fragment 1-85 is
believed to have an anti-inflammatory effect against inflammation
induced by LPS (lipopolysaccharide) and such. Wei Gong et al. have
reported that a fragment of 1-85 fused with fragment 186-215
reduces the rate of death caused by LPS administration more than
fragment 1-85 (Journal of Biomedicine and Biotechnology Volume
2010, Article ID 915234, doi: 10.1155/2010/915234). The article of
Wei Gong et al. suggests that the acidic tail is required to
increase the anti-inflammatory effect of 1-85. On the other hand,
the present invention demonstrated that the acidic tail rather
inhibited the migration of bone marrow mesenchymal stem cells. In
view of the present invention, it is expected that shortening or
complete elimination of the acidic tail can achieve more
improvement of therapeutic effects on diseases on which
administration of bone marrow mesenchymal stem cells has
therapeutic effects.
EXAMPLE 18
Methods
[0719] Experimental animals used were SD rats (male, eight weeks
old). After sufficiently deep anesthesia by isoflurane inhalation,
a rectangular skin incision of 3 cm width.times.7 cm length was
made on the back. The cephalic side was left uncut, and the skin
was thoroughly detached from the subcutaneous tissues. The three
incised sides were sutured to the surrounding skin using #4 silk
suture, and protected with Tegaderm (3M) to prevent bacterial
infection.
[0720] The full-length mouse HMGB1 (100 .mu.g/administration/day)
produced in HEK293 and chemically synthesized HMGB1 peptide (amino
acids 1 to 44; 50 .mu.g/administration/day) were diluted to 200
.mu.l with phosphate buffer and administered to rats via the caudal
vein. The first administration was performed 6 hours after the
surgery, and then the agents were administered every 24 hours a
total of five times. Phosphate-buffered physiological saline was
administered as a negative control. Tegaderm was removed after one
week, and the wound region was observed weekly to measure the areas
of necrosis and ulceration.
Results
[0721] One week after surgery, skin necrosis developed in four of
five rats of the negative control group. In the group administered
with the full-length HMGB1, skin necrosis developed in three of
five rats. In the HMGB1 peptide (amino acids 1-44) group, skin
necrosis developed in one of five rats. Seven weeks after surgery,
while severe skin contracture occurred in four of five rats of the
negative control group, three of five rats of the full-length
HMGB1-administered group and two of five rats of the HMGB1 peptide
(amino acids 1-44) group showed such contracture (FIG. 24).
Discussion
[0722] The shrinking effect on necrotizing tissues after one week
was observed in the group administered with the full-length HMGB1
produced in HEK293 and in the HMGB1 peptide (amino acids 1-44)
administration group. The shrinking effect was stronger in the
HMGB1 peptide (amino acids 1-44) group. After one, two, and three
weeks, the wound area was reduced to a half of the area of the
negative control group. After the third week, the wound area in the
HMGB1 peptide (amino acids 1-44) administration group was further
reduced as compared to the other two groups. During the 7-week
healing process, there was also a tendency that the wound area was
smaller in the HMGB1 peptide (amino acids 1-44) group. The
contracture at the wound site after seven weeks was also the
mildest in the HMGB1 peptide (amino acids 1-44) group. Bone marrow
mesenchymal stem cells are known to enhance the growth of skin
cells under low-oxygen conditions. It is presumed that bone marrow
mesenchymal stem cells recruited by HMGB1 enhanced the wound
healing by suppressing the expansion of skin necrosis due to
undernutrition and hypoxia caused by skin flap production. It is
suggested that such effects are advantageous not only in
suppressing expansion of damage caused by skin ischemia, injury,
and surgery but also in the cosmetic aspect after healing.
EXAMPLE 19
Methods
[0723] Experimental animals used were 15 SD rats (male, eight weeks
old) per group. After sufficient anesthesia by isoflurane
inhalation, a rectangular skin incision of 3 cm width.times.7 cm
length was made on the back. The cephalic side was left uncut, and
the skin was thoroughly detached from the subcutaneous tissues. The
incised three sides were sutured to the surrounding skin using #4
silk suture, and protected with Tegaderm (3M) to prevent bacterial
infection.
[0724] Chemically synthesized HMGB1 peptide (amino acids 1-44; 50
.mu.g/administration/day) or HMGB1 peptide (amino acids 17-25; 50
.mu.g/administration/day) was diluted to 200 .mu.l with phosphate
buffer and administered to the rats with experimental wound. The
first administration was performed 6 hours after the surgery, and
then the agents were administered every 24 hours a total of five
times. Phosphate-buffered physiological saline was administered as
a negative control. Tegaderm was removed after one week, and the
wound was observed two weeks after the wound production. Uncured
areas (ulceration and necrosis) were measured.
Results
[0725] The ratio (%) of the uncured area relative to the area of
the whole skin flap after two weeks of the skin flap production was
calculated. The uncured area accounted for 14.1% on average in the
HMGB1 peptide (amino acids 1-44) group and 9.1% on average in the
HMGB1 peptide (amino acids 17 to 25, 50 .mu.g/administration/day)
group. In addition, the ratio (%) of the uncured area relative to
the area of the whole skin flap after six weeks of the skin flap
production was 4.1% on average in the HMGB1 peptide (amino acids
1-44) group and 3.5% on average in the HMGB1 peptide (amino acids
17-25; 50 .mu.g/administration/day) group (FIG. 26).
Discussion
[0726] In the test using the skin damage model in Example 19, there
was a tendency that the wound area during the healing process from
weeks 1 to 7 after damage was smaller in the group administered
with chemically synthesized HMGB1 peptide (amino acids 1-44) as
compared to the group administered with the full-length HMGB1
produced in HEK293. Furthermore, the test of the present Example
demonstrated that the skin damage-improving effect observed in the
chemically-synthesized HMGB1 peptide (amino acids 17-25) group was
comparable to or greater than that seen in the HMGB1 peptide (amino
acids 1-44, 50 .mu.g/administration/day) group. There are multiple
regions (core sequences) having the activity of recruiting bone
marrow mesenchymal stem cells in vitro. Of these, the shortest
sequence known at present is the sequence consisting of 9 amino
acids from position 17 to 25. The other core sequences with the
activity of recruiting mesenchymal stem cells require 30 amino
acids or 85 amino acids in length. The experiment of this Example
demonstrated that the group administered with the HMGB1 peptide
consisting of only 9 amino acids (amino acids 17-25) showed
improvement of the skin damage not only in vitro but also in vivo.
Peptides containing the 9 amino acids as the core domain are
expected to be much more stably produced at a lower cost as
compared to protein preparations produced from eukaryote-derived
cultured cells such as HEK293.
INDUSTRIAL APPLICABILITY
[0727] The present invention provides peptides retaining activity
of recruiting PDGFR.alpha.-positive cells, whose molecular weights
are, for example, one tenth or less as compared to the full-length
HMGB1 protein consisting of about 200 amino acids. Such peptides
can be produced by chemical synthesis methods using peptide
synthesizers, as well as production methods using E. coli or
eukaryote-derived cultured cells. Thus, when the peptides are
produced as pharmaceuticals, one can expect purity improvement,
stable production, and cost reduction.
[0728] Furthermore, when the recombinant peptides were produced in
E. coli or cultured cells, they showed improved activity that was
about twice or about six times that of the full-length HMGB1 when
compared per mole or per mass, respectively. Thus, when the
peptides are clinically used as pharmaceuticals, it is possible to
use reduced dosages. This leads to cost reduction and prevention of
side effects.
[0729] In addition, the full-length HMGB1 is known to have the
binding activity with lipopolysaccharide (LPS), which is an
endotoxin. Furthermore, there is a report that HMGB1 fragments of
amino acids 1 to 79 or amino acids 88 to 162 of HMGB1 lack the
LPS-binding activity (Youn et al., J Immunol 2008,
180;5067-5074).
[0730] When pharmaceuticals are contaminated with even a trace
amount of LPS, they cause fever and such, often resulting in severe
adverse effects. Therefore, there are strict regulations against
contamination of pharmaceuticals with LPS. Since HMGB1 has affinity
for LPS, it is difficult to completely remove contaminating LPS
from pharmaceuticals. However, since the conversion into peptides
results in reduction of the affinity for LPS, it is expected that
contamination of pharmaceuticals with LPS can be reduced. Thus, by
using peptides consisting of a portion identified in the present
invention as recruiting PDGFR.alpha.-positive cells, it is possible
to develop much safer pharmaceuticals.
[0731] By administering a peptide of the present invention to a
tissue in need of regeneration or an adjacent tissue thereof,
regeneration of the tissue can be induced or promoted. Moreover, by
administering a peptide of the present invention to a tissue other
than a tissue in need of regeneration by intravenous administration
and such, regeneration of the tissue in need of regeneration can be
induced or promoted. For example, in the treatment of a disease of
deep-seated organ, such as cerebral infarction, it is difficult to
administer a therapeutic agent directly to a damaged site (brain).
On the other hand, in the present invention, such treatment can be
carried out by intravenous administration, which is widely used in
general medical practice. It is therefore possible to administer a
therapeutic agent at any concentration and frequency in a safe and
simple manner. This is a superior effect as compared to
conventional therapeutic methods.
[0732] Meanwhile, a recently developed bone marrow cell-based
method that is known to be effective in treating cerebral
infarction involves the collection of cells from patient's bone
marrow and re-administration of the cells into the bloodstream.
This method is inevitably associated with severe invasion because
bone marrow cells need to be aspirated with a large-bore needle
inserted into the bone marrow, which is located deep inside the
body. In contrast, the present invention allows bone marrow cells
to be recruited directly to the bloodstream by intravenous
administration of an agent, and therefore does not involve severe
invasion even when the agent is frequently administered to cerebral
infarction patients.
[0733] Bone marrow-derived pluripotent stem cells have the
potential ability to differentiate into various types of cells such
as mesenchymal cells, epithelial cells, and nerve cells. After
migrating to a damaged site, they may differentiate depending on a
niche environment surrounding the damaged site, and then induce
tissue repair. In regenerative medicine and cell therapy, bone
marrow pluripotent stem cells, which are rare cells, are expanded
by ex vivo culture before use in the treatment. However, this
requires adequate safety control because, unlike conventional
pharmaceutical agents, there is a risk of deterioration of cells
(canceration and contamination with bacteria, viruses, etc.) which
may be caused during the culturing process. On the other hand, the
present invention is highly safe because the cells are not removed
from the body for artificial manipulation.
Sequence CWU 1
1
141215PRTHomo sapiens 1Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly
Lys Met Ser Ser Tyr1 5 10 15Ala Phe Phe Val Gln Thr Cys Arg Glu Glu
His Lys Lys Lys His Pro 20 25 30Asp Ala Ser Val Asn Phe Ser Glu Phe
Ser Lys Lys Cys Ser Glu Arg 35 40 45Trp Lys Thr Met Ser Ala Lys Glu
Lys Gly Lys Phe Glu Asp Met Ala 50 55 60Lys Ala Asp Lys Ala Arg Tyr
Glu Arg Glu Met Lys Thr Tyr Ile Pro65 70 75 80Pro Lys Gly Glu Thr
Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95Arg Pro Pro Ser
Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110Ile Lys
Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys 115 120
125Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp
Ile Ala145 150 155 160Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala
Lys Lys Gly Val Val 165 170 175Lys Ala Glu Lys Ser Lys Lys Lys Lys
Glu Glu Glu Glu Asp Glu Glu 180 185 190Asp Glu Glu Asp Glu Glu Glu
Glu Glu Asp Glu Glu Asp Glu Asp Glu 195 200 205Glu Glu Asp Asp Asp
Asp Glu 210 2152648DNAHomo sapiens 2atgggcaaag gagatcctaa
gaagccgaga ggcaaaatgt catcatatgc attttttgtg 60caaacttgtc gggaggagca
taagaagaag cacccagatg cttcagtcaa cttctcagag 120ttttctaaga
agtgctcaga gaggtggaag accatgtctg ctaaagagaa aggaaaattt
180gaagatatgg caaaagcgga caaggcccgt tatgaaagag aaatgaaaac
ctatatccct 240cccaaagggg agacaaaaaa gaagttcaag gatcccaatg
cacccaagag gcctccttcg 300gccttcttcc tcttctgctc tgagtatcgc
ccaaaaatca aaggagaaca tcctggcctg 360tccattggtg atgttgcgaa
gaaactggga gagatgtgga ataacactgc tgcagatgac 420aagcagcctt
atgaaaagaa ggctgcgaag ctgaaggaaa aatacgaaaa ggatattgct
480gcatatcgag ctaaaggaaa gcctgatgca gcaaaaaagg gagttgtcaa
ggctgaaaaa 540agcaagaaaa agaaggaaga ggaggaagat gaggaagatg
aagaggatga ggaggaggag 600gaagatgaag aagatgaaga tgaagaagaa
gatgatgatg atgaataa 6483215PRTMus musculus 3Met Gly Lys Gly Asp Pro
Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr1 5 10 15Ala Phe Phe Val Gln
Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30Asp Ala Ser Val
Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45Trp Lys Thr
Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60Lys Ala
Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro65 70 75
80Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro
Lys 100 105 110Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val
Ala Lys Lys 115 120 125Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp
Asp Lys Gln Pro Tyr 130 135 140Glu Lys Lys Ala Ala Lys Leu Lys Glu
Lys Tyr Glu Lys Asp Ile Ala145 150 155 160Ala Tyr Arg Ala Lys Gly
Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175Lys Ala Glu Lys
Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu 180 185 190Asp Glu
Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu 195 200
205Glu Glu Asp Asp Asp Asp Glu 210 2154648DNAMus musculus
4atgggcaaag gagatcctaa aaagccgaga ggcaaaatgt cctcatatgc attctttgtg
60caaacttgcc gggaggagca caagaagaag cacccggatg cttctgtcaa cttctcagag
120ttctccaaga agtgctcaga gaggtggaag accatgtctg ctaaagaaaa
ggggaaattt 180gaagatatgg caaaggctga caaggctcgt tatgaaagag
aaatgaaaac ctacatcccc 240cccaaagggg agaccaaaaa gaagttcaag
gaccccaatg cacccaagag gcctccttcg 300gccttcttct tgttctgttc
tgagtaccgc cccaaaatca aaggcgagca tcctggctta 360tccattggtg
atgttgcaaa gaaactagga gagatgtgga acaacactgc agcagatgac
420aagcagccct atgagaagaa agctgccaag ctgaaggaga agtatgagaa
ggatattgct 480gcctacagag ctaaaggaaa acctgatgca gcgaaaaagg
gggtggtcaa ggctgaaaag 540agcaagaaaa agaaggaaga ggaagatgat
gaggaggatg aagaggatga ggaagaggag 600gaagaagagg aagacgaaga
tgaagaagaa gatgatgatg atgaataa 6485215PRTRattus norvegicus 5Met Gly
Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr1 5 10 15Ala
Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25
30Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
Ala 50 55 60Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr
Ile Pro65 70 75 80Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro
Asn Ala Pro Lys 85 90 95Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser
Glu Tyr Arg Pro Lys 100 105 110Ile Lys Gly Glu His Pro Gly Leu Ser
Ile Gly Asp Val Ala Lys Lys 115 120 125Leu Gly Glu Met Trp Asn Asn
Thr Ala Ala Asp Asp Lys Gln Pro Tyr 130 135 140Glu Lys Lys Ala Ala
Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala145 150 155 160Ala Tyr
Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170
175Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu
180 185 190Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu
Asp Glu 195 200 205Glu Glu Asp Asp Asp Asp Glu 210 2156648DNARattus
norvegicus 6atgggcaaag gagatcctaa gaagccgaga ggcaaaatgt cctcatatgc
attctttgtg 60caaacctgcc gggaggagca caagaagaag cacccggatg cttctgtcaa
cttctcagag 120ttctccaaga agtgctcaga gaggtggaag accatgtctg
ctaaagaaaa ggggaaattt 180gaagatatgg caaaggctga caaggctcgt
tatgaaagag aaatgaaaac ctacatcccc 240cccaaagggg agaccaaaaa
gaagttcaag gaccccaatg cccccaagag gcctccttcg 300gccttcttct
tgttctgttc tgagtaccgc ccaaaaatca aaggcgagca tcctggctta
360tccattggtg atgttgcgaa gaaactagga gagatgtgga acaacactgc
tgcggatgac 420aagcagccct atgaaaagaa ggccgccaag ctgaaggaga
agtatgagaa ggatattgct 480gcctacagag ctaaaggaaa acctgatgca
gcgaaaaagg gggtggtcaa ggctgagaag 540agcaagaaaa agaaggaaga
ggaagacgac gaggaggatg aagaggatga ggaagaggag 600gaagaggagg
aagacgaaga tgaagaagaa gatgatgatg atgaataa
64875PRTArtificialfragment added to the expressed protein or
peptides 7Gly Pro Gly Tyr Gln1 5860DNAArtificialnucleotide sequence
of cloning site of the pCAGGS vectorCDS(1)..(60) 8cat cac cat cac
cat cac tcc gcg gct ctt gaa gtc ctc ttt cag gga 48His His His His
His His Ser Ala Ala Leu Glu Val Leu Phe Gln Gly1 5 10 15ccc ggg tac
cag 60Pro Gly Tyr Gln 20920PRTArtificialamino acid sequence of
cloning site of the pCAGGS vector 9His His His His His His Ser Ala
Ala Leu Glu Val Leu Phe Gln Gly1 5 10 15Pro Gly Tyr Gln
201063DNAArtificialartificial nucleotide sequence coding secretory
signal sequence 10atggagacag acacactcct gctatgggta ctgctgctct
gggttccagg ttccactggt 60gac 631114PRTArtificialartificial 6xHis-tag
and TEV protease recognition site 11Met Lys His His His His His His
Glu Asn Leu Tyr Phe Gln1 5 10126PRTArtificial6xHis-tag 12His His
His His His His1 5137PRTArtificialTEV protease recognition site
13Glu Asn Leu Tyr Phe Gln Gly1 514228PRTArtificialrecombinant
hsHMGB1 with 6xHis-tag and TEV protease recognition site 14Met Lys
His His His His His His Glu Asn Leu Tyr Phe Gln Gly Lys1 5 10 15Gly
Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe 20 25
30Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser
35 40 45Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys
Thr 50 55 60Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys
Ala Asp65 70 75 80Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile
Pro Pro Lys Gly 85 90 95Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala
Pro Lys Arg Pro Pro 100 105 110Ser Ala Phe Phe Leu Phe Cys Ser Glu
Tyr Arg Pro Lys Ile Lys Gly 115 120 125Glu His Pro Gly Leu Ser Ile
Gly Asp Val Ala Lys Lys Leu Gly Glu 130 135 140Met Trp Asn Asn Thr
Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys145 150 155 160Ala Ala
Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg 165 170
175Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu
180 185 190Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu Asp
Glu Glu 195 200 205Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp
Glu Glu Glu Asp 210 215 220Asp Asp Asp Glu225
* * * * *