U.S. patent application number 11/041363 was filed with the patent office on 2005-10-06 for segment of glycosylation-deficient hgf alpha-chain.
Invention is credited to Fukuta, Kazuhiro, Matsumoto, Kunio, Nakamura, Toshikazu.
Application Number | 20050221441 11/041363 |
Document ID | / |
Family ID | 34650773 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050221441 |
Kind Code |
A1 |
Nakamura, Toshikazu ; et
al. |
October 6, 2005 |
Segment of glycosylation-deficient HGF alpha-chain
Abstract
The present invention provides a segment of
glycosylation-deficient HGF having mutation(s) introduced into an
amino acid sequence so as to prevent glycosylation at least one
glycosylation site of a hepatocyte growth factor (HGF), and a
method of producing the same. The segment of
glycosylation-deficient HGF of the present invention has the same
activity as that of a segment of glycosylated HGF, therefore, it is
useful as an alternate for a segment of glycosylated HGF.
Inventors: |
Nakamura, Toshikazu; (Kyoto,
JP) ; Matsumoto, Kunio; (Mino, JP) ; Fukuta,
Kazuhiro; (Mino, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
34650773 |
Appl. No.: |
11/041363 |
Filed: |
January 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11041363 |
Jan 25, 2005 |
|
|
|
10926088 |
Aug 26, 2004 |
|
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Current U.S.
Class: |
435/69.4 ;
435/320.1; 435/325; 530/399; 536/23.5 |
Current CPC
Class: |
C07K 14/4753
20130101 |
Class at
Publication: |
435/069.4 ;
435/320.1; 435/325; 530/399; 536/023.5 |
International
Class: |
C07K 014/475; C07H
021/04; C12N 015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2004 |
JP |
018882/2004 |
Claims
1. A segment of glycosylation-deficient HGF, having a deficiency of
at least one sugar chain at glycosylation site(s) of an HGF .alpha.
chain, and selected from the following proteins (a) to (c), wherein
the above-mentioned protein has an N terminal hairpin domain and
the subsequent four kringle domains, has an antagonist activity
against the action of HGF via a c-Met/HGF receptor, and has a
neovascularization inhibitory activity: (a) a protein having an
amino acid sequence from 32nd to 494th in SEQ ID NO: 1, (b) a
protein having an amino acid sequence from 32nd to 494th of SEQ ID
No. 1, wherein 1 to 30 amino acids are deleted, substituted, added
or inserted in said amino acid sequence, (c) a protein having a
homology of at least 80% or higher with amino acids from 32nd to
494th of SEQ ID NO: 1.
2. The segment of glycosylation-deficient HGF according to claim 1,
wherein the amino acid(s) is/are substituted so as to prevent
glycosylation of at least one glycosylation site of the
protein.
3. The segment of glycosylation-deficient HGF according to claim 2,
wherein the substitution(s) of amino acid(s) is/are at least one of
the following (a) to (d): (a) Asn of at least one N-glycosylation
site is substituted by another amino acid, wherein N-glycosylation
site means a portion having consensus sequence represented by the
amino acid sequence Asn-X-Ser or Asn-X-Thr wherein X represents an
amino acid other than Pro, (b) Ser or Thr of one N-glycosylation
site, or Ser and/or Thr of two or more N-glycosylation sites,
is/are substituted by another amino acid, wherein N-glycosylation
site means a portion having consensus sequence represented by the
amino acid sequence Asn-X-Ser or Asn-X-Thr wherein X represents an
amino acid other than Pro, (c) X of at least one N-glycosylation
site is substituted by Pro, wherein N-glycosylation site means a
portion having consensus sequence represented by the amino acid
sequence Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid
other than Pro, and (d) at least one of Ser and Thr which undergoes
O-glycosylation is substituted by another amino acid.
4. The segment of glycosylation-deficient HGF according to claim 2,
wherein the substitution(s) of amino acid(s) is/are at least one of
the following (a) to (d): (a) amino acid(s) at 294th and/or 296th
of an amino acid sequence of SEQ ID NO: 1 is/are substituted by
another amino acid, and/or an amino acid at 295th thereof is
substituted by Pro, leading thereby to no glycosylation at 294th,
(b) amino acid(s) at 402nd and/or 404th of an amino acid sequence
of SEQ ID NO: 1 is/are substituted by another amino acid, and/or an
amino acid at 403rd thereof is substituted by Pro, leading thereby
to no glycosylation at 402nd, or (c) an amino acid at 476th of an
amino acid sequence of SEQ ID NO: 1 is substituted by another amino
acid, leading thereby to no glycosylation at 476th.
5. The segment of glycosylation-deficient HGF according to claim 1,
wherein amino acids at 162nd to 166th of an amino acid sequence of
SEQ ID NO: 1 are deleted.
6. The segment of glycosylation-deficient HGF according to claim 1,
wherein amino acids at 479th to 494th of an amino acid sequence of
SEQ ID NO: 1 are deleted.
7. A DNA comprising a base sequence coding the segment of
glycosylation-deficient HGF according to claim 1.
8. A vector containing the DNA according to claim 7 incorporated
therein.
9. A method of producing the segment of glycosylation-deficient HGF
according to claim 1, comprising introducing a vector containing a
DNA comprising a base sequence coding the segment of
glycosylation-deficient HGF according to claim 1 into a cell,
culturing the cell, allowing a segment of glycosylation-deficient
HGF to be accumulated in the cell or to be secreted into culture
solution of the cell, and collecting and purifying the segment of
glycosylation-deficient HGF from the cell or culture solution of
the cell.
10. The method of producing the segment of glycosylation-deficient
HGF according to claim 9, wherein the cell is a eukaryotic
cell.
11. The method of producing the segment of glycosylation-deficient
HGF according to claim 10, wherein the eukaryotic cell is a yeast
or insect cell.
12. A method of producing the segment of glycosylation-deficient
HGF according to claim 1, comprising introducing a vector
containing a DNA comprising a base sequence coding the segment of
glycosylation-deficient HGF according to claim 1 into an insect
individual, allowing a segment of glycosylation-deficient HGF to be
accumulated in the insect individual, and collecting and purifying
the segment of glycosylation-deficient HGF from the insect
individual.
13. A method of producing the segment of glycosylation-deficient
HGF according to claim 1, comprising treating a segment of HGF
having sugar chain(s) with an enzyme to remove the sugar chain(s)
wholly or partially, and collecting and purifying the segment of
glycosylation-deficient HGF from the enzymatic reaction
mixture.
14. A method of producing the segment of glycosylation-deficient
HGF according to claim 1, comprising introducing a vector
containing an incorporated DNA coding a segment of HGF having sugar
chain(s) or a vector containing a DNA comprising a base sequence
coding the segment of glycosylation-deficient HGF according to
claim 1 into a cell having no glycosylation ability, culturing the
cell, allowing a segment of glycosylation-deficient HGF to be
accumulated in the cell or to be secreted into culture solution of
the cell, and collecting and purifying the segment of
glycosylation-deficient HGF from the cell or culture solution of
the cell.
15. A method of producing the segment of glycosylation-deficient
HGF according to claim 1, comprising synthesizing a segment of
glycosylation-deficient HGF by cell-free protein synthesis system
using a DNA having a base sequence coding a segment of HGF having
sugar chain(s) or a DNA comprising a base sequence coding the
segment of glycosylation-deficient HGF according to claim 1 as a
template, and collecting and purifying the segment of
glycosylation-deficient HGF from the synthesis reaction
mixture.
16. A method of producing the segment of glycosylation-deficient
HGF according to claim 1, comprising limited digestion of
glycosylation-deficient HGF which lacks sugar chain(s) at at least
one or all of glycosylation sites, by treatment with a protease or
by chemical treatment of HGF, and collecting and purifying the
segment of glycosylation-deficient HGF from the treatment
solution.
17. The method according to claim 16, wherein the protease is
esterase.
18. An agent comprising as an active ingredient the segment of
glycosylation-deficient HGF according to claim 1.
19. The agent according to claim 18, wherein the agent is a
neovascularization inhibiting agent.
20. The agent according to claim 18, wherein the agent is an
antagonist against the action of HGF mediated via a c-Met/HGF
receptor.
21. The agent according to claim 18, wherein the agent is an agent
suppressing invasion, growth or metastasis of tumor.
22. The agent according to claim 18, wherein the agent is an
apoptosis-inducing agent.
23. The agent according claim 20, wherein the agent is an agent for
prevention and/or treatment of an infectious disease.
24. The agent according claim 23, wherein the infectious disease is
an infectious disease caused by a malaria or Listeria parasite.
25. A gene pharmaceutical composition, comprising the DNA according
to claim 7.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a modified product of a
segment of HGF (hepatocyte growth factor) used for preventing and
treating cancers or diseases caused by excessive
neovascularization. More specifically, the present invention
relates to a segment of HGF, modified by deficiency of
glycosylation.
[0003] 2. Description of the Related Art
[0004] For therapy of cancers, surgical therapy, chemotherapy,
radiation therapy or multidisciplinary therapy combining them and
the like are conducted, however, remarkable anticancer methods are
not established yet. Though a primary tumor can be removed by
surgical therapy, there is often formation of invisible small
metastatic cancers when the cancer is found in the case of cancers
showing a tendency of metastasis such as pancreatic and lung
cancers, even if the size of the primary tumor is small, and
surgical removal thereof is difficult. In chemotherapy and
radiation therapy using an anticancer agent, it is difficult to
prevent recurrence of a resistant cancer and metastasis of survived
cancer cells although the primary tumor may be temporarily
decreased in size. With an anticancer agent and radiation therapy,
killing also normal cells in addition to cancer cells is
accompanied by severe side effects, resulting in decrease in the
quality of life and immune power of a patient. Thus, there is
currently no method of efficiently preventing cancer metastasis,
and there is a strong desire for development of drugs effective for
preventing cancer metastasis.
[0005] There are a lot of researches conducted in the past
regarding the mechanisms of cancer metastasis. Many cancers develop
in epithelial tissue, and cancer cells are released from a primary
tumor, break a basement membrane partitioning epithelial tissue,
invade into surrounding tissues, enter blood and lymphatic vessels
and are carried to distal tissues by blood and lymph flow, and
again manifest invasion into tissues from blood and lymphatic
vessels and growth therein associated with neovascularization.
[0006] For suppressing cancer metastasis, it is believed
advantageous to inhibit any of these processes, and listed as
cancer metastasis suppressing agents are, for example, substances
suppressing adhesion of cancer cells with vascular endothelial
cells at metastasis site (see non-patent literature 1),
neovascularization inhibitors (see non-patent literature 2),
substances suppressing invasion of cancer cells (see patent
literature 1), substances inhibiting enzymes which degrade basement
membranes (see patent literature 2, non-patent literature 3) and
the like.
[0007] Recently, tumor dormancy therapy is attracting attention
regarding cancer therapy. The tumor dormancy therapy makes a
dormant condition of cancer cells using a neovascularization
inhibitor. The neovascularization inhibitor does not directly kill
cancer cells but inhibits neovascularization to block a route for
feeding oxygen and nutrition necessary for growth of cancer cells,
which results in inducing apoptosis and making a dormant condition
of cancer cells. Known as the neovascularization inhibitor are
Angiostatin (see non-patent literature 2), Endostatin (see
non-patent literature 4), and the like.
[0008] Neovascularization occurs by proliferation of capillary
vessels of preexisting blood vessels. Neovascularization is
indispensable for many physiological processes such as
embryogenesis, wound healing, tissue and organ regeneration, while
abnormal neovascularization occurs under pathogenic conditions such
as tumor growth and at metastatic tumor sites. Initiation of tumor
neovascularization is known to be induced by vascular endothelial
cell growth factor (VEGF), basic fibroblast growth factor (bFGF),
HGF (HGF; Hepatocyte growth factor) and the like.
[0009] In addition to such a neovascularization action, HGF has a
mitogenic activity, motogenic activity and morphogenic activity,
and originally works as a regeneration factor supporting natural
healing power of a living body. It is known that various biological
activities of HGF are expressed through binding of HGF to a
c-Met/HGF receptor on a target cell. The c-Met/HGF receptor is
known to be often excessively expressed in many cancer cells (see
non-patent literatures 5 to 7), and HGF induces invasion and
metastasis of tumor cells (see non-patent literature 8). Cancer
cells invade and metastasize by utilizing the actions of HGF owned
by a living body through mutual interactions with interstitial
cells surrounding the cancer cells (see non-patent literatures 8,
9). Therefore, it is supposed that if binding of HGF to a c-Met/HGF
receptor on tumor cells is inhibited, invasion and metastasis of
the tumor cells can be suppressed.
[0010] NK4 is a protein composed of an N-terminal hairpin domain
and four kringle domains of an .alpha.-chain of HGF. NK4 binds to a
c-Met/HGF receptor and acts as an antagonist against HGF (see
non-patent literatures 10, 11). NK4 is known to suppress invasion
and metastasis of a tumor by an HGF antagonist activity. Further,
NK4 is known to suppress neovascularization induced not only by HGF
but also by VEGF and bFGF by a mechanism other than an HGF
antagonist activity (see non-patent literature 12). Thus, NK4 is a
bi-functional molecule having an HGF antagonist activity and a
neovascularization inhibitory activity simultaneously, and NK4 is
expected to be utilized as a novel anticancer agent.
[0011] Further, NK4 can be expected, based on its
neovascularization inhibitory activity, to be also utilized as an
agent for preventing or treating various diseases ascribable to
blood vessel abnormal proliferation, for example, rheumatic
arthritis, diabetic retinopathy, immature infant retinopathy,
senile macular degeneration, excess scar formation in wound healing
and the like. As described above, neovascularization is
indispensable for maintaining metabolism of tissues under normal
condition and keeping functional homeostasis of a living body,
however, it is known that abnormal neovascularization is related
with various diseases including inflammatory diseases. For example,
it has been reported that metastasis and recurrence of solid tumors
and diseases such as proliferative diabetes, psoriasis vulgaris,
rheumatic arthritis, diabetic retinopathy, senile macular
degeneration, excess scar formation in wound healing are ascribable
to abnormal proliferation of blood vessels, particularly,
peripheral capillary vessels (see non-patent literatures 13 and
14). NK4 having a neovascularization inhibitory activity is
promising as an agent for preventing or treating such diseases.
[0012] Further, NK4 can be expected to provide application as a
drug for infection prevention and treatment of Listeria and
malaria. It is known that a c-Met/HGF receptor is used as a base
for human infection with Listeria monocytogenes (see non-patent
literature 15). Furthermore, activation of a c-Met/HGF receptor by
HGF is known to be essential for the initial mechanism of human
infection with malaria parasite (see non-patent literature 16).
Therefore, it is believed that NK4, an antagonist against HGF,
manifests an effect of prevention and treatment of these infectious
diseases.
[0013] It is necessary to mass-produce an NK4 protein using cells
by genetic engineering methods for use, as a pharmaceutical
preparation, of an NK4 protein exerting an effect of prevention and
treatment of various diseases, as described above. Conventionally,
it is known that NK4 can be produced using animal cells such as
Chinese hamster ovary (CHO) cells (see patent literature 3),
however, in general, methods of producing a protein using animal
cells such as CHO cells are expensive, indicating resultant
increase in drug price.
[0014] As a method of producing a recombinant protein at low cost,
known is a method of introducing the intended gene into a
prokaryote such as E. coli and allowing this to express (see
non-patent literature 17). However, there is a problem that a sugar
chain cannot be added to the recombinant protein produced in a
prokaryote such as E. coli. The reason for this is that a
prokaryote such as E. coli does not contain an endoplasmic
reticulum and Golgi apparatus which are places for biosynthesis of
a sugar chain.
[0015] Addition of a sugar chain to a protein and its modification
in an animal cell is post-translational modification using no
template, differing from the case of biosynthesis of DNA or
proteins. This post-translational modification is conducted through
a complicated mechanism via a lot of glycosylation-related enzymes
locally present in cell organellas called endoplasmic reticulum and
Golgi apparatus. That is, according to a complicated biosynthetic
pathway catalyzed by enzymes (glycosidases and
glycosyltransferases) specific to certain linkages of
monosaccharides, a sugar chain is elongated so as to obtain a given
structure while being subjected to sequential cutting and addition
of monosaccharides (see non-patent literature 18). It is known that
sugar chains thus added to proteins are widely involved in whole
life phenomenon in higher organisms (see non-patent literatures 19,
20).
[0016] It is known that half or more of proteins in a human body
are present in the form of a glycoprotein carrying sugar chains
(see non-patent literature 21), and if a glycoprotein originally
present in the form carrying sugar chains is converted into a form
containing no sugar chain, there is a fear of losing activity. For
example, it is known that erythropoietin known as an erythropoietic
hormone loses pharmaceutical activities when sugar chains are
removed (see non-patent literature 22).
[0017] Yeast is known as a cell which is a host capable of
producing a recombinant protein at low cost and having a
glycosylation ability (see non-patent literatures 23 to 25). Since
yeast is a eukaryote and has an endoplasmic reticulum and Golgi
apparatus, it is consequently equipped with glycosylation
mechanism. However, since the glycosylation mechanism of yeast
differs significantly from that of animal cells, when a protein
having glycosylation site(s) is produced in yeast, sugar chain(s)
of yeast type would be added. The sugar chain structures of yeast
differ significantly from those of other mammals (see non-patent
literature 26), and such a recombinant protein manifests
antigenicity against human and other mammals, therefore, it cannot
be used as a medicament for human and animals.
[0018] Further, an insect cell is also a host having a
glycosylation ability and can produce a protein at relatively low
cost, however, the sugar chain structures of an insect cell are
also different from those of human type (see non-patent literature
27), and there is a possibility for a recombinant protein derived
from insect cells to show antigenicity against human and other
mammals.
[0019] Then, one can envisage production of a protein containing no
sugar chains by removal of sugar chains from a protein produced
using yeast and insect cells and the like, or by introduction of a
gene designed to have mutation(s) at glycosylation sites in a
protein molecule into yeast and insect cells and the like. However,
if a protein originally present in the form carrying sugar chains
is converted into a protein containing no sugar chain, there is a
fear of losing activity, as described above.
[0020] NK4 is a fragment of an HGF .alpha.-chain and contains three
glycosylation sites of an HGF .alpha.-chain (see non-patent
literatures 28 and 29). There is utterly no information regarding
whether or not NK4 has an activity when sugar chains are removed
from NK4.
[0021] (Patent Literature 1)
[0022] JP-A No. 3-31214
[0023] (Patent Literature 2)
[0024] JP-A No. 5-194414
[0025] (Patent Literature 3)
[0026] JP-A No. 2003-250549
[0027] (Non-Patent Literature 1)
[0028] Iwamoto Y, et al., Science, 1987, p. 1132-1134
[0029] (Non-Patent Literature 2)
[0030] Cao Y, et al., The Journal of clinical investigation, 1988,
vol. 101, p. 1055-1063
[0031] (Non-Patent Literature 3)
[0032] Irimura T, et al., Biochemistry, 1989, vol. 25, p.
5322-5328
[0033] (Non-Patent Literature 4)
[0034] Blezinger P, et al., Nature Biotechnology, 1999, vol. 17, p.
343-348
[0035] (Non-Patent Literature 5)
[0036] Di Renzo, et al., Oncogene, 1991, vol. 6, p. 1997-2003
[0037] (Non-Patent Literature 6)
[0038] Di Renzo, et al., Cancer research, 1995, vol. 55, p.
1129-1138
[0039] (Non-Patent Literature 7)
[0040] Nakajima M., et al., Cancer, 1999, vol. 85, p. 1894-1902
(1999)
[0041] (Non-Patent Literature 8)
[0042] Jiang W. G., et al., Critical reviews in
oncology/hematology, 1999, vol. 29, p. 209-248
[0043] (Non-Patent Literature 9)
[0044] Nakamura T., et al., Cancer research, 1997, vol. 57, p.
3305-3313
[0045] (Non-Patent Literature 10)
[0046] Date K., et al., FEBS letters, 1997, vol. 420, p. 1-6
[0047] (Non-Patent Literature 11)
[0048] Date K., et al., Oncogene, 1998, vol. 17, p. 3045-3054
[0049] (Non-Patent Literature 12)
[0050] Kuba K., et al., Cancer research, 2000, vol. 60, p.
6737-6743
[0051] (Non-Patent Literature 13)
[0052] Polverini P J., Critical reviews in oral biology and
medicine, 1995, vol. 6 (no. 3), p. 230-247
[0053] (Non-Patent Literature 14)
[0054] Forkman J., Nature medicine, 1995, vol. 1 (no. 1), p.
27-31
[0055] (Non-Patent Literature 15)
[0056] Shen Y., et al., Cell, 2000, vol. 103, p. 501-510
[0057] (Non-Patent Literature 16)
[0058] Carrolo M., et al., Nature Med, 2003, vol. 9, p.
1363-1369
[0059] (Non-Patent Literature 17)
[0060] Swarts J. R., Current opinion in biotechnology, 2001, vol.
12, p. 195-201
[0061] (Non-Patent Literature 18)
[0062] Kornfeld R., et al., Annual review of biochemistry, 1985,
vol. 54, p. 631-664
[0063] (Non-Patent Literature 19)
[0064] Kobata A., European journal of biochemistry, 1992, vol. 209,
p. 483-501
[0065] (Non-Patent Literature 20)
[0066] Varki A., Glycobiology, 1993, vol. 3, p. 97-130
[0067] (Non-Patent Literature 21)
[0068] Goochee C. F., et al., Biotechnology, 1991, vol. 9, p.
1347-1355
[0069] (Non-Patent Literature 22)
[0070] Takeuchi M., et al., Glycobiology, 1991, vol. 1, p.
337-346
[0071] (Non-Patent Literature 23)
[0072] Wiseman A., Endeavour, 1996, vol. 20, p. 130-132
[0073] (Non-Patent Literature 24)
[0074] Russell C., et al., Australian journal of biotechnology,
1991, vol. 5, p. 48-55
[0075] (Non-Patent Literature 25)
[0076] Buckholz R. G., et al., Biotechnology, 1991, vol. 9, p.
1067-1072
[0077] (Non-Patent Literature 26)
[0078] Gemmill T. R., et al., Biochimica et biophysica acta, 1999,
vol. 1426, p. 227-237
[0079] (Non-Patent Literature 27)
[0080] Altmann F., et al., Glycoconjugate journal, 1999, vol. 16,
p. 109-123
[0081] (Non-Patent Literature 28)
[0082] Hara H., et al., Journal of biochemistry, 1993, vol. 114, p.
76-82
[0083] (Non-Patent Literature 29)
[0084] Shimizu N., et al., Biochemical and biophysical research
communications, 1992, vol. 189, p. 1329-1335
SUMMARY OF THE INVENTION
[0085] An object of the present invention is to provide a segment
of HGF, particularly an HGF .alpha.-chain, and an intramolecular
fragment of an HGF .alpha.-chain, which are modified by
glycosylation-deficiency, and a method of producing the same.
[0086] The present inventors have intensively studied the sugar
chain function of an NK4 protein to solve the above-mentioned
problems, and resultantly found that the function of NK4 is kept
even if sugar chains of an NK4 protein are removed. It was utterly
unexpected that an NK4 protein which is a bi-functional molecule
having an HGF antagonist activity and a neovascularization
inhibitory activity simultaneously keeps these functions
equivalently to glycosylated NK4 even if sugar chains are removed.
Based on the above-mentioned findings, the present inventors have
further developed investigations and completed the present
inventions.
[0087] Namely, the present invention relates to
[0088] (1) A segment of glycosylation-deficient HGF, having a
deficiency of at least one sugar chain at glycosylation sites of an
HGF .alpha. chain, and selected from the following proteins (a) to
(c), wherein the above-mentioned protein has an N terminal hairpin
domain and the subsequent four kringle domains, has an antagonist
activity against the action of HGF via a c-Met/HGF receptor, and
has a neovascularization inhibitory activity:
[0089] (a) a protein having an amino acid sequence from 32nd to
494th in SEQ ID NO: 1,
[0090] (b) a protein having an amino acid sequence from 32nd to
494th of SEQ ID No. 1, wherein 1 to 30 amino acids are deleted,
substituted, added or inserted in said amino acid sequence,
[0091] (c) a protein having a homology of at least 80% or higher
with amino acids from 32nd to 494th of SEQ ID NO: 1,
[0092] (2) The segment of glycosylation-deficient HGF according to
(1), wherein the amino acid(s) is/are substituted so as to prevent
glycosylation at least one glycosylation site of the protein,
[0093] (3) The segment of glycosylation-deficient HGF according to
(2), wherein the substitution(s) of amino acid(s) is/are at least
one of the following (a) to (d):
[0094] (a) Asn of at least one N-glycosylation site is substituted
by another amino acid, wherein N-glycosylation site means a portion
having consensus sequence represented by the amino acid sequence
Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid other
than Pro.
[0095] (b) Ser or Thr of one N-glycosylation site, or Ser and/or
Thr of two or more N-glycosylation sites, is/are substituted by
another amino acid, wherein N-glycosylation site means a portion
having consensus sequence represented by the amino acid sequence
Asn-X-Ser or Asn-X-Thr wherein X represents an amino acid other
than Pro.
[0096] (c) X of at least one N-glycosylation site is substituted by
Pro, wherein N-glycosylation site means a portion having consensus
sequence represented by the amino acid sequence Asn-X-Ser or
Asn-X-Thr wherein X represents an amino acid other than Pro,
and
[0097] (d) at least one of Ser and Thr which undergoes
O-glycosylation is substituted by another amino acid.
[0098] (4) The segment of glycosylation-deficient HGF according to
(2), wherein the substitution(s) of amino acid(s) is/are at least
one of the following (a) to (d):
[0099] (a) amino acid(s) at 294th and/or 296th of an amino acid
sequence of SEQ ID NO: 1 is/are substituted by another amino acid,
and/or an amino acid at 295th thereof is substituted by Pro,
leading thereby to no glycosylation at 294th,
[0100] (b) amino acid(s) at 402nd and/or 404th of an amino acid
sequence of SEQ ID NO: 1 is/are substituted by another amino acid,
and/or an amino acid at 403rd thereof is substituted by Pro,
leading thereby to no glycosylation at 402nd, or
[0101] (c) an amino acid at 476th of an amino acid sequence of SEQ
ID NO: 1 is substituted by another amino acid, leading thereby to
no glycosylation at 476th,
[0102] (5) The segment of glycosylation-deficient HGF according to
any one of (1) to (4), wherein amino acids at 162nd to 166th of an
amino acid sequence of SEQ ID NO: 1 are deleted, and
[0103] (6) The segment of glycosylation-deficient HGF according to
any one of (1) to (5), wherein amino acids at 479th to 494th of an
amino acid sequence of SEQ ID NO: 1 are deleted.
[0104] Further, the present invention relates to
[0105] (7) A DNA comprising a base sequence coding the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
[0106] (8) A vector containing the DNA according to (7)
incorporated therein,
[0107] (9) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising introducing the vector according to (8) into a cell,
culturing the cell, allowing a segment of glycosylation-deficient
HGF to be accumulated in the cell or to be secreted into culture
solution of the cell, and collecting and purifying the segment of
glycosylation-deficient HGF from the cell or culture solution of
the cell,
[0108] (10) The method of producing the segment of
glycosylation-deficient HGF according to (9), wherein the cell is a
eukaryotic cell,
[0109] (11) The method of producing the segment of
glycosylation-deficient HGF according to (10), wherein the
eukaryotic cell is a yeast or insect cell,
[0110] (12) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising introducing the vector according to (8) into an insect
individual, allowing a segment of glycosylation-deficient HGF to be
accumulated in the insect individual, and collecting and purifying
the segment of glycosylation-deficient HGF from the insect
individual,
[0111] (13) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising treating a segment of HGF having sugar chain(s) with an
enzyme to remove the sugar chain(s) wholly or partially, and
collecting and purifying the segment of glycosylation-deficient HGF
from the enzymatic reaction mixture,
[0112] (14) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising introducing a vector containing an incorporated DNA
coding a segment of HGF having sugar chain(s) or the vector
according to (8) into a cell having no glycosylation ability,
culturing the cell, allowing a segment of glycosylation-deficient
HGF to be accumulated in the cell or to be secreted into culture
solution of the cell, and collecting and purifying the segment of
glycosylation-deficient HGF from the cell or culture solution of
the cell,
[0113] (15) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising synthesizing a segment of glycosylation-deficient HGF by
cell-free protein synthesis system using a DNA having a base
sequence coding a segment of HGF having sugar chain(s) or the DNA
according to (7) as a template, and collecting and purifying the
segment of glycosylation-deficient HGF from the synthesis reaction
mixture,
[0114] (16) A method of producing the segment of
glycosylation-deficient HGF according to any one of (1) to (6),
comprising limited digestion of glycosylation-deficient HGF which
lacks at least one or all of glycosylation sites, by treatment with
a protease or by chemical treatment of HGF, and collecting and
purifying the segment of glycosylation-deficient HGF from the
treatment solution, and
[0115] (17) The method of production according to (16), wherein the
protease is an esterase.
[0116] Still further, the present invention relates to
[0117] (18) An agent comprising the segment of
glycosylation-deficient HGF according to any one of (1) to (6) as
an active ingredient,
[0118] (19) The agent according to (18), wherein the agent is a
neovascularization inhibiting agent,
[0119] (20) The agent according to (18), wherein the agent is an
antagonist against the action of HGF via a c-Met/HGF receptor,
[0120] (21) The agent according to any one of (18) to (20), wherein
the agent is an agent suppressing invasion, growth or metastasis of
a tumor,
[0121] (22) The agent according to anyone of (18) to (20), wherein
the agent is an apoptosis inducing agent,
[0122] (23) The agent according (20), wherein the agent is an agent
for prevention and/or treatment of an infectious disease,
[0123] (24) The agent according (23), wherein the infectious
disease is an infectious disease caused by a malaria or Listeria
parasite, and
[0124] (25) A gene pharmaceutical composition, comprising the DNA
according to (7).
[0125] Furthermore, the present invention relates to use of a
segment of a glycosylation-deficient HGF for producing a
pharmaceutical preparation for inhibiting neovascularization and
the like, and a method of preventing and treating
neovascularization and the like by administrating an agent
containing a segment of a glycosylation-deficient HGF as an active
ingredient to a patient. Also, the present invention relates to a
use of DNA coding a segment of a glycosylation-deficient HGF for
producing a gene pharmaceutical composition for inhibiting
neovascularization and the like, and a method of preventing and
treating neovascularization and the like by administrating a gene
pharmaceutical composition containing DNA coding a segment of a
glycosylation-deficient HGF to a patient.
[0126] The segment of a glycosylation-deficient HGF of the present
invention (hereinafter, abbreviated as segment of
glycosylation-deficient HGF) can be produced by the use of yeast or
insect cells, and therefore, a segment of a glycosylation-deficient
HGF can be produced at low cost. Particularly, it is significant to
be able to produce glycosylation-deficient NK4 at low cost.
[0127] Since the segment of a glycosylation-deficient HGF of the
present invention (for example, glycosylation-deficient NK4) is
equivalent to a segment of HGF having sugar chain(s) (for example,
NK4 having sugar chain(s)) with regard to an HGF antagonist
activity, neovascularization inhibitory activity and the like, it
can be an alternate for a segment of HGF having sugar chain(s) (for
example, NK4 having sugar chain(s)). That is, the segment of a
glycosylation-deficient HGF of the present invention can be used
for suppressing invasion, suppressing growth, suppressing
metastasis, inducing apoptosis and/or suppressing
neovascularization of tumor cells. An agent containing DNA coding a
segment of a glycosylation-deficient HGF of the present invention
can be used as a gene pharmaceutical composition.
BRIEF DESCRIPTION OF THE DRAWING
[0128] FIG. 1 is a view showing results of analysis of glycosylated
NK4 and glycosylation-deficient NK4 by SDS-PAGE.
[0129] FIG. 2 is a view showing the action of NK4 suppressing cell
migration induced by HGF, compared between glycosylated NK4 and
glycosylation-deficient NK4.
[0130] FIG. 3 is a view showing the action of NK4 suppressing the
proliferation of vascular endothelial cells induced by bFGF,
compared between glycosylated NK4 and glycosylation-deficient
NK4.
[0131] FIG. 4 is a view showing the action of NK4 suppressing the
invasion of cancer cells, compared between glycosylated NK4 and
glycosylation-deficient NK4.
[0132] FIG. 5 is a view showing results of Western blot analysis of
glycosylated NK4 and glycosylation-deficient NK4 produced by pichia
pastoris.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0133] The present invention will be illustrated in detail
below.
[0134] In the present invention, "a segment of a
glycosylation-deficient HGF" means a segment of HGF in which all or
at least one sugar chain at glycosylation sites of an HGF
.alpha.-chain, preferably all sugar chains, are deficient. "Segment
of HGF" means, for example, an .alpha.-chain protein of HGF of
human and mammals (for example, dogs, cats, cattle, horses, pigs,
sheep, etc.), having an amino acid sequence in 32nd to 494th of SEQ
ID NO: 1 and having an N-terminal hairpin domain and the subsequent
four kringle domain structures, or a protein of an intramolecular
fragment of HGF having the above-mentioned N-terminal hairpin
domain and the subsequent four kringle domains, preferably a
protein of an intramolecular fragment of an HGF .alpha.-chain. As
the protein of an intramolecular fragment of an HGF .alpha.-chain,
listed are proteins obtained from an HGF .alpha.-chain (for
example, 32nd to 494th of SEQ ID NO: 1) by deletion of 1 to 30,
preferably 1 to 25, more preferably 1 to 21 amino acids.
Specifically listed is a 5 amino acid-deleted type HGF
.alpha.-chain represented by 32nd to 489th of SEQ ID NO: 2, being
obtained by deletion of 162nd to 166th amino acids of SEQ ID NO: 1,
for example. Further listed are NK4 represented by an amino acid
sequence in 32nd to 478th of SEQ ID NO: 1 (479th to 494th amino
acids are deleted in an amino acid sequence represented by SEQ ID
NO: 1), and NK4 represented by an amino acid sequence in 32nd to
473rd of SEQ ID NO: 2 (474th to 489th amino acids are deleted in an
amino acid sequence represented by SEQ ID NO: 2), for example.
[0135] In the segment of a glycosylation-deficient HGF of the
present invention, the protein having an amino acid sequence
obtained from an HGF .alpha.-chain (for example, 32nd to 494th of
SEQ ID NO: 1) by deletion, substitution, addition or insertion of 1
to 30, preferably 1 to 25 amino acids means a protein obtained by
deletion, substitution, addition or insertion of amino acids by
genetic engineering methods and/or spontaneous mutation and the
like. Specifically mentioned are proteins of an intramolecular
fragment of an HGF .alpha.-chain described above, proteins obtained
by addition of amino acid(s) to N-terminal and/or C-terminal of an
HGF .alpha.-chain, and/or proteins obtained by deletion,
substitution, addition or insertion and the like of amino acid(s)
of glycosylation site(s) of an HGF .alpha.-chain by genetic
engineering methods so as to inhibit glycosylation. Proteins
obtained by deletion, substitution, addition or insertion and the
like of amino acid(s) by using well-known techniques such as
site-directed mutagenesis methods, and proteins naturally produced
by deletion, substitution, addition or insertion and the like of
amino acid(s) of a number 1 to several are also included.
[0136] Also included are proteins having a homology of 80% or more,
preferably 85% or more, more preferably 90% or more with an amino
acid sequence of an HGF .alpha.-chain in a segment of a
glycosylation-deficient HGF of the present invention. In the
present invention, "homology" means the extent of coincidence of
amino acid residues constituting respective sequences when primary
structures of proteins are compared.
[0137] Those having a modified amino acid residue, pyroglutamic
acid, as the N-terminal amino acid of a protein are also included
in the segment of a glycosylation-deficient HGF of the present
invention.
[0138] The segment of a glycosylation-deficient HGF of the present
invention is a protein having an antagonist activity against the
action of HGF mediated via a c-Met/HGF receptor and having a
neovascularization inhibitory activity. Listed as the action of HGF
mediated via a c-Met/HGF receptor are a tyrosine phosphorylation of
a c-Met/HGF receptor, motogenic activity, mitogenic activity and
morphogenic activity (Matsumoto K. and Nakamura T., Biochem.
Biophys. Res. Commun., 1997, vol. 239, p. 639-644). The
neovascularization inhibiting action preferably means an action of
inhibiting neovascularization in which HGF, FGF and VEGF are
involved though its mechanism is not critical when it is an action
inhibiting neovascularization.
[0139] Deficiency of sugar chain(s) of a segment of HGF can be
generated by introducing mutation(s) into a base sequence to cause
deletion, substitution and addition of amino acid(s) in an amino
acid sequence so as to prevent addition of sugar chain(s)
preferably at least one of glycosylation sites of an HGF
.alpha.-chain, however, deficiency can also be caused by treating a
segment of HGF having sugar chain(s) with some enzymes.
[0140] As the method of introducing mutation(s) into a gene of a
segment of HGF so as to prevent addition of sugar chain(s) at least
one of glycosylation sites of an HGF .alpha.-chain, it is
advantageous to introduce mutation(s) into a base sequence coding
glycosylation site to be made deficient.
[0141] Two types of sugar chains are known. One is an N-linked type
sugar chain and the other is an O-linked type sugar chain. The
following mutations are introduced into a base sequence,
respectively.
[0142] In the case of the N-linked type sugar chain, a consensus
sequence (Asn-X-Ser or Asn-X-Thr (X represents an amino acid other
than proline)) subjected to glycosylation is known. In the case of
the presence of a consensus sequence, a sugar chain is added to Asn
in the consensus sequence (Kobata A., Eur. J. Biochem., 1992, vol.
209, p. 483-501). Therefore, an N-linked type sugar chain can be
made deficient by introducing mutation(s) into a base sequence so
as to convert Asn in a consensus sequence into another amino acid
(for example, Gln and the like), or to convert Ser or Thr in the
consensus sequence into another amino acid (for example, Gly, Ala
and the like). In this case, it is preferable to appropriately
select an amino acid for conversion so as not to form a new
consensus sequence with backward and forward amino acid sequences
of the above-mentioned consensus sequence. It may also be
permissible to introduce mutation(s) into a base sequence so that
proline is introduced at X site in a consensus sequence.
[0143] In the case of the O-linked type sugar chain, a sugar chain
is added to a hydroxyl group of Ser or Thr in an O-glycosylation
site, however, a consensus sequence for O-glycosylation does not
exist. A sugar chain at the O-glycosylation site can be made
deficient by introducing mutation(s) into a base sequence so as to
convert Ser or Thr subjected to O-glycosylation into another amino
acid (for example, Gly and the like). In this case, it is
preferable to appropriately select an amino acid for conversion so
as not to form a consensus sequence for N-glycosylation with
backward and forward amino acid sequences of the amino acid.
[0144] Glycosylation-deficient site(s) of a segment of a
glycosylation-deficient HGF coincides with glycosylation site(s) of
a segment of HGF, and in NK4 as a segment of an intramolecular
fragment of an .alpha.-chain of human HGF having an amino acid
sequence from 32nd to 478th of SEQ ID NO: 1 for example, they are
Asn (N-linked type) at 294th, Asn (N-linked type) at 402nd and Thr
(O-linked type) at 476th. Glycosylation site(s) in NK4 as an
intramolecular fragment of an .alpha.-chain of 5 amino acid-deleted
human HGF having an amino acid sequence from 32nd to 473rd of SEQ
ID NO: 2 (hereinafter, abbreviated as 5 amino acid-deleted NK4)
is/are Asn (N-linked type) at 289th, Asn (N-binding type) at 397th
and Thr (O-linked type) at 471st of SEQ ID NO.:2. The
above-mentioned consensus sequence for N-glycosylation in the
above-mentioned NK4 is present at from 294th to 296th and from
402nd to 404th of an amino acid sequence of SEQ ID NO: 1, and in
the 5 amino acid-deleted NK4, the consensus sequence for
N-glycosylation is present at from 289th to 291st and from 397th to
399th in an amino acid sequence of SEQ ID NO: 2.
[0145] Regarding the method of introducing mutation(s) into a base
sequence, when, for example, mutations are introduced into DNA
coding NK4, mutagenic primers corresponding to a portion into which
mutations are to be introduced are synthesized, and introduction
can be conducted using a known technology such as a Kunkel method.
By using a commercially available mutagenesis kit and the like,
mutations can be introduced easily. It is also possible that based
on known base sequence information on NK4, a base sequence at
portions into which mutations are to be introduced is modified, and
DNA is produced by chemical synthesis using a conventionally known
method. As the chemical synthesis method, there are listed chemical
synthesis methods using DNA synthesizers such as a DNA synthesizer
model 392 (manufactured by Perkin Elmer) utilizing a
phosphoramidite method.
[0146] DNA coding a segment of an HGF .alpha.-chain, for example,
DNA coding NK4, can be obtained, for example, by a method described
in JP-A No. 2003-250549 or according to this method. For example,
primers are prepared based on a known HGF base sequence, and DNA is
amplified by a PCR method using, as a template, HGF cDNA
synthesized from HGF mRNA contained in human tissues or cells or
HGF cDNA selected from cDNA library, thereby enabling to obtain DNA
coding NK4. It is also possible to amplify cDNA by a RT-PCR method
from mRNA contained in human tissues or cells. Further, it can be
obtained also by the above-mentioned chemical synthesis.
[0147] A recombinant expression vector for a segment of
glycosylation-deficient HGF can be constructed from a recombinant
vector such as a plasmid and phage containing DNA coding an amino
acid sequence of a segment of a glycosylation-deficient HGF, by
excising this DNA with a restriction enzyme, and re-connecting it
to downstream of a promoter of a vector suitable for expression of
a segment of a glycosylation-deficient HGF by using a restriction
enzyme and DNA ligase. More specifically, construction is so made
that it contains, if necessary, (1) promoter, (2) ribosome binding
site, (3) initiation codon, (4) DNA containing a base sequence
coding a segment of a glycosylation-deficient HGF of the present
invention, (5) termination codon and (6) terminator in this order
toward downstream direction of transcription.
[0148] By incorporating a secretion signal sequence before a base
sequence coding a segment of a glycosylation-deficient HGF, the
segment of a glycosylation-deficient HGF can be secreted out of a
cell. The signal sequence is preferably to be recognized by a host
cell and processed. For example, when the host cell belongs to
Escherichia species, signal sequences of PhoA, OmpA and the like
can be utilized, when the host cell belongs to Bacillus species,
signal sequences of .alpha.-amylase, subtilisin and the like can be
utilized, when the host cell is yeast, signal sequences of
MF.alpha., SUC2 and the like can be utilized, and when the host
cell is a mammalian cell, signal sequences of HGF, insulin,
.alpha.-interferon and the like can be utilized, respectively. In a
segment of human HGF, it is particularly preferable to utilize a
signal sequence of human HGF. Specifically, a base sequence coding
an amino acid sequence 1 to 31 as depicted in SEQ ID NO: 1
corresponds to a signal sequence.
[0149] The above-mentioned DNA includes not only DNA composed of a
base sequence coding a sequence of a segment of
glycosylation-deficient HGF obtained by introducing mutation(s)
into the above-mentioned base sequence coding glycosylation
site(s), but also (a) DNA having a base sequence having deletion,
substitution, addition or insertion of one or more bases in a base
sequence coding the above-mentioned sequence of a segment of
glycosylation-deficient HGF having an HGF antagonist activity and
neovascularization inhibitory activity, (b) DNA hybridizing under
stringent conditions with DNA composed of a base sequence
complimentary to DNA having a base sequence coding the
above-mentioned sequence of a segment of a glycosylation-deficient
HGF having an HGF antagonist activity and neovascularization
inhibitory activity, (c) DNA having a homology of at least 80% or
more with DNA having a base sequence coding the above-mentioned
sequence of a segment of glycosylation-deficient HGF having an HGF
antagonist activity and neovascularization inhibitory activity.
[0150] "Deletion, substitution, addition or insertion of one or
more bases" regarding the above-mentioned base sequences means
deletion, substitution, addition or insertion and the like of bases
of a number 1 to several, which can be introduced by well-known
technological methods such as a site-directed mutagenesis method or
can be introduced naturally.
[0151] DNA hybridizable under stringent conditions means DNA
obtained by a colony hybridization method, plaque hybridization
method, southern blot hybridization method and the like using the
above-mentioned DNA as a probe.
[0152] The stringent conditions mean hybridization conditions, for
example, SSC solution of about 0.1 to 2-fold concentration (SSC
solution at 1-fold concentration contains 150 mM sodium chloride
and 15 mM sodium citrate) at temperatures of about 65.degree.
C.
[0153] DNA having homology means DNA showing a homology of at least
about 80% or more under high stringent conditions, preferably DNA
having a homology of about 85% or more, more preferably DNA having
a homology of about 90% or more. The high stringent conditions
include, for example, a sodium concentration of about 19 to 40 mM,
preferably about 19 to 20 mM, and a temperature of about 50 to
70.degree. C., preferably about 60 to 65.degree. C. Particularly, a
sodium concentration of about 19 mM and a temperature of about
65.degree. C. are most preferable conditions.
[0154] As the vector which can be used in the present invention,
plasmids such as pBR 322, pUC18, pUC19 (Toyobo Co. Ltd.) can be
used in the case of Escherichia coli as a host, plasmids such as
pUB110 (Sigma) can be used in the case of Bacillus subtilis as a
host, and plasmids such as pYES2 (Invitrogen), pRB15 (ATCC 37062)
can be used in the case of yeast as a host. As the expression
vector for animal cells, listed are pCAGGS and pCXN2 (Niwa H.,
Yamamura K. and Miyazaki J., Gene, 1991, vol. 108, p. 193 to 200,
JP-A No. 03-168087), pcDL-SR.alpha. (Takebe Y., et al., Mol. Cell.
Biol., 1988, vol. 8, p. 466-472) and the like. Additionally,
bacteriophage .lambda.gt10, .lambda.gt11 (Stratagene), vector
derived from a gene of SV40 (BRL), BPV (ATCC VR-703), retrovirus
and the like are listed, however, there is no specific restriction
providing they are vectors capable of replicating and amplifying in
a host.
[0155] Also regarding promoters and terminators, there is no
specific restriction so long as they work in a host used for
expression of a base sequence coding an intended segment of a
glycosylation-deficient HGF. As the promoter, listed are trp
promoter, lac promoter, recA promoter, .lambda.PL promoter, lpp
promoter and the like in the case of Escherichia coli as a host,
and listed are PHO5 promoter, PGK promoter, GAP promoter, ADH
promoter and the like in the case of yeast as a host. When animal
cells are used as a host, promoters obtained from virus genomes
such as Rous sarcoma virus (virus RSV), MPSV, polyoma virus,
fowlpox virus, adenovirus, bovine papilloma virus, fowl sarcoma
virus, cytomegalovirus (CMV), hepatitis B virus, simian virus 40
(SV40), and vaccinia virus; metallo-thioneine promoter; heat shock
promoter; and the like are listed. In the case of using a higher
mammal host, an enhancer is preferably introduced into a vector. By
introducing an enhancer, transcription increases. Listed as the
enhancer are SV40 enhancer, initial promoter/enhancer of
cytomegalovirus, polyoma enhancer, adenovirus enhancer and the
like. As the terminator, listed are trp terminator, lpp terminator
and the like in the case of Escherichia coli as a host, listed are
amyF terminator and the like in the case of Bacillus subtilis as a
host, listed are CYC1 terminator and the like in the case of yeast
as a host, and listed are SV40 terminator, HSV1TK terminator and
the like in the case of animal cells as a host. These promoters and
terminators are appropriately combined depending on the host
used.
[0156] The cell which can be used as a host is not particularly
restricted, and eukaryotic cells such as animals, plants, insects,
eukaryotic microorganisms and the like, and prokaryotic cells such
as prokaryotic microorganisms and the like are listed. These cells
may form an individual, and animal individuals, plant individuals
and insect individuals may be used as a host. The eukaryotic cell
may be an adherent cell or floating cell, for example, may be a
eukaryotic cell producing and accumulating a segment of HGF in the
cell, or a eukaryotic cell producing and secreting a segment of HGF
out of the cell. As the animal cell, for example, CHO cell (Chinese
hamster ovary cell), COS cell, BHK cell, mouse C127 cell and Hela
cell and the like are listed. As the plant cell, for example, cells
of rice, tobacco, Arabidopsis thaliana and the like are listed, and
as the insect cell, for example, cells of Sf9, Sf21 and the like
are listed. As the insect individual, for example, silk worm
(Bombyx mori) is mentioned. As the eukaryotic microorganism, yeasts
such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Candida boidinii, Pichia pastoris and the like, and filamentous
fungi such as Aspergillus, Trichoderma, Mucor and the like are
listed. As the prokaryotic microorganism, Escherichia coli and
Bacillus subtilis and the like are listed, and these prokaryotic
cells have no glycosylation ability, therefore, an expression
vector coding a segment of HGF containing glycosylation sites may
be introduced. Preferable are yeasts, insect cells or insect
individuals.
[0157] An expression vector for a segment of a
glycosylation-deficient HGF is introduced into a host by a
competent cell method (J. Mol. Biol., 1970, vol. 53, p. 154),
protoplast method (Proc. Natl. Acad. Sci. USA, 1978, vol. 75, p.
1929), calcium phosphate method (Science, 1983, vol. 221, p. 551),
DEAE dextran method (Science, 1982, vol. 215, p. 166), electric
pulse method (Proc. Natl. Acad. Sci. USA, 1984, vol. 81, p. 7161),
in vitro packaging method (Proc. Nat. Acad. Sci. USA, 1975, vol.
72, p. 581), virus vector method (Cell, 1984, vol. 37, p. 1053),
micro injection method (Exp. Cell. Res., 1984, vol. 153, p. 347)
and the like, to produce a transformant. The resultant transformant
is cultured in an appropriate medium corresponding to its host for
the purpose of producing an intended segment of a
glycosylation-deficient HGF. The medium contains carbon sources,
nitrogen sources, inorganic substances, vitamins, serum and
medicaments and the like necessary for growth of the transformant.
As the medium, LB medium (Nissui Pharmaceutical Co., Ltd.), M9
medium (J. Exp. Mol. Genet., Cold Spring Laboratory, New York,
1972, p. 431) and the like are listed when the host of a
transformant is Escherichia coli, and YEPD medium (Genetic
Engineering, vol. 1, Plenum Press, New York, 1979, p. 117) and the
like are listed when the host is yeast. When the host is an animal
cell, MEM medium, DMEM medium, RPMI 1640 medium (Nissui
Pharmaceutical Co., Ltd.) containing 20% or less fetal bovine
serum, and the like are listed. Culturing of a transformant is
conducted usually at a temperature of 20 to 45.degree. C. and a pH
of 5 to 8, and ventilation and stirring are conducted as required.
When the host is animal adherent cell and the like, carriers such
as glass beads, collagen beads or acetyl cellulose hollow fiber are
used. Culturing of a transformant can be conducted even with a
medium composition or under culturing conditions other than the
above compositions and conditions so long as the transformant can
grow, therefore, the composition and culturing condition are not
limited to the above-mentioned examples.
[0158] In culturing, when a protease inhibitor is added into
culture solution, decomposition of a segment of a
glycosylation-deficient HGF can be suppressed, leading to
improvement in productivity of the segment of a
glycosylation-deficient HGF. As the protease inhibitor to be added,
benzamidine, aprotinin, leupeptin, antipain, chymostatin, pepstatin
A, phenylmethanesulfonyl fluoride (PMSF), ethylenediamine
tetraacetic acid (EDTA) and the like are listed.
[0159] The segment of a glycosylation-deficient HGF thus produced
in a transformant or the culture supernatant of a transformant can
be separated, collected and purified by a known salting out method,
solvent precipitation method, dialysis method, ultrafiltration
method, gel electrophoresis method or gel filtration
chromatography, ion exchange chromatography, reverse phase
chromatography, affinity chromatography and the like in
combination. Particularly, combinations of a salting out method
using ammonium sulfate, S-sepharose ion chromatography, heparin
sepharose affinity chromatography and phenylsepharose hydrophobic
chromatography, or combinations of a salting out method using
ammonium sulfate, S-sepharose ion chromatography and anti-HGF
antibody sepharose affinity chromatography, and the like are
preferable and effective purification methods.
[0160] The segment of a glycosylation-deficient HGF of the present
invention can also be prepared by obtaining a segment of
glycosylated HGF by conventionally known methods, then, treating
the segment of HGF with an enzyme removing a sugar chain. As the
enzyme removing a sugar chain, there can be used glycopeptidase F,
glycopeptidase A and the like for the purpose of removing an
N-linked type sugar chain. Removal of an O-linked type sugar chain
can be attained by combining sialidase, fucosidase, O-glycanase and
the like. In this case, it is desirable to add a protease inhibitor
into the reaction solution. As the protease inhibitor to be added,
benzamidine, aprotinin, leupeptin, antipain, chymostatin, pepstatin
A, phenylmethanesulfonyl fluoride (PMSF), ethylenediamine
tetraacetic acid (EDTA) and the like are listed. The segment of HGF
from which a sugar chain is removed by enzymatic treatment can be
collected as the segment of a glycosylation-deficient HGF of the
present invention and purified by the above-mentioned purification
method.
[0161] Further, the segment of a glycosylation-deficient HGF of the
present invention can be obtained also by utilizing cell-free
protein synthesis system. The cell-free protein synthesis system
means a method of performing protein synthesis using DNA or mRNA
coding the intended protein as a template without using a live
cell, while using cell extract prepared from Escherichia coli,
rabbit reticulocyte, wheat germ and the like or using protein
synthesis factors contained in the cell extract solution. Since
cell extract solution contains molecules necessary for protein
synthesis such as ribosome, tRNA, and translation factor, a protein
is synthesized upon addition of an energy source such as ATP, GTP,
etc. and amino acids as substrates. Instead of cell extract
solution, a mixture of protein synthesis factors contained in cell
extract solution may be used. In the cell-free protein synthesis
system, a segment of a glycosylation-deficient HGF can be produced
using, as a template, DNA or mRNA coding a segment of HGF having
glycosylation site(s), because an endoplasmic reticulum and Golgi
apparatus are not contained therein. DNA or mRNA having mutation(s)
introduced into glycosylation site(s) can also be used.
[0162] A protease inhibitor can also be added into the reaction
mixture. As the protease inhibitor to be added, benzamidine,
aprotinin, leupeptin, antipain, chymostatin, pepstatin A,
phenylmethanesulfonyl fluoride (PMSF), ethylenediamine tetraacetic
acid (EDTA) and the like are listed. The segment of a
glycosylation-deficient HGF synthesized in the cell-free protein
synthesis reaction mixture can be collected and purified by the
above-mentioned purification method.
[0163] The segment of a glycosylation-deficient HGF of the present
invention can be produced also by digesting glycosylation-deficient
HGF with a protease, or by specifically cutting this by chemical
treatment. Glycosylation-deficient HGF used for this purpose can be
produced by introducing mutation(s) into glycosylation site(s) of
HGF based on an already known gene sequence of HGF. Enzymatic
digestion of glycosylation-deficient HGF can be conducted using a
specific protease such as, for example, elastase, HGF activator,
urokinase type plasminogen activator, matriptase, etc. The segment
of a glycosylation-deficient HGF generated can be collected and
purified by the above-mentioned purification method.
[0164] The segment of a glycosylation-deficient HGF of the present
invention obtained as described above has an activity equivalent to
that of the segment of a glycosylated HGF in an antagonist activity
against the action of HGF mediated via a c-Met/HGF receptor and a
neovascularization inhibitory activity.
[0165] The segment of a glycosylation-deficient HGF of the present
invention is effective as a pharmaceutical product, and used in the
form of general pharmaceutical preparation. The pharmaceutical
preparation containing the segment of a glycosylation-deficient HGF
of the present invention as an active ingredient can adopt various
dosage forms (for example, liquid, solid, capsule and the like),
and in general, the segment of a glycosylation-deficient HGF as an
active ingredient is used in combination with a pharmaceutically
acceptable carrier to give an injection, inhalant, suppository or
oral agent, and an injection is suitable. This injection can be
prepared by a normal method, and for example, can be prepared by
dissolving a segment of a glycosylation-deficient HGF and a
pharmaceutically acceptable carrier into a suitable solvent (for
example, sterile purified water, buffer solution, physiological
saline solution and the like), then, filtrating the solution
through a filter and the like for sterilization, then, filling this
in a sterile vessel. The amount of a segment of a
glycosylation-deficient HGF in an injection is usually adjusted
from about 0.0002 to 3 (w/v %), preferably from 0.001 to 2 (w/v %).
The oral drug is formulated into a dosage form such as, for
example, tablet, granule, fine granule, powder, soft or hard
capsule, liquid, emulsion, suspension, syrup and the like, and
these preparations can be prepared by an ordinary method for
preparation. The suppository can also be prepared by an ordinary
method for preparation using a conventional base (for example,
cacao butter, lauric butter, glycerogelatine, Macrogol, Witepsol
and the like). The inhalant can also be prepared according to
normal means for preparation. The amount of a segment of a
glycosylation-deficient HGF in a preparation can be appropriately
adjusted depending on dosage form, disease to be treated and the
like.
[0166] In the formulation of a pharmaceutical preparation of the
segment of a glycosylation-deficient HGF of the present invention,
a stabilizer is preferably added. As the stabilizer, for example,
albumin, globulin, gelatin, alanine, glycine, mannitol, glucose,
dextran, sorbitol, ethylene glycol and the like are listed. The
pharmaceutical preparation of the present invention may contain
necessary other additives, for example, solvents (for example,
physiological saline solution, sterile purified water, injectable
water and the like), excipients (for example, fructose, D-sorbitol,
glucose, starch, crystalline cellulose, dextrin and the like),
binders (for example, gelatin, corn starch, tragacanth, gum arabic
and the like), solubilizers (for example, lauromacrogol,
Polysorbate 80, polyoxyethylene hardened castor oil 60, gum arabic,
sodium benzoate and the like), antioxidants (for example,
L-ascorbic acid, tocopherol, sodium edetate and the like), soothing
agents (for example, benzalkonium chloride, procaine hydrochloride
and the like), isotonic agents (for example, sodium chloride,
glucose, D-mannitol, glycerin and the like), buffers (for example,
citric acid, sodium citrate, acetic acid, sodium acetate, lactic
acid, sodium hydrogenphosphate and the like), thickening agents
(gum arabic, carmellose, popidone, methylcellulose and the like),
preservatives (for example, methyl p-oxybenzoate, ethyl
p-oxybenzoate, propyl p-oxybenzoate, chlorobutanol, benzyl alcohol,
benzalkonium chloride and the like), pH adjusters (hydrochloric
acid, sodium hydroxide, citric acid, acetic acid and the like), and
the like.
[0167] In the case of liquid preparation, it is preferable to
retain the preparation by cryopreservation, or by lyophilization
and the like to remove moisture. In the case of a lyophilized
preparation, injectable distilled water and the like are added
before its use to reconstitute the preparation.
[0168] In the case of oral preparation, it is preferable to apply a
film of an enteric coating agent (for example, cellulose acetate
phthalate, methacrylic acid copolymer, hydroxypropylcellulose
phthalate, carboxymethylethyl cellulose and the like) and the like
to make a granule, tablet and the like, and in the case of capsule,
an enteric coated capsule is preferable.
[0169] The preparation of the present invention can be administered
via a suitable administration route corresponding to its dosage
form. For example, it can be made into a form of injection and
administered intravenously, intraarterially, subcutaneously,
intramuscularly, etc. The dose thereof is appropriately adjusted
depending on disease, symptom, age, body weight and the like of a
patient, and for example, it is from 0.01 mg to 500 mg, preferably
from 0.05 mg to 100 mg in adults in the case of a segment of a
glycosylation-deficient HGF (for example, glycosylation-deficient
NK4), and once daily administration or several times daily
administration is suitable.
[0170] The segment of a glycosylation-deficient HGF of the present
invention can be used for suppressing invasion, suppressing growth,
suppressing metastasis, inducing apoptosis and/or inhibiting
neovascularization of tumor. Therefore, an agent containing the
segment of a glycosylation-deficient HGF of the present invention
can be used as an agent for preventing and treating a cancer and/or
a disease caused by neovascularization, and an agent for preventing
metastasis of cancer.
[0171] Examples of the target cancers include lung cancer, ovarian
cancer, pancreatic cancer, stomach cancer, gallbladder cancer,
kidney cancer, prostatic cancer, breast cancer, esophageal cancer,
liver cancer, oral cavity cancer, colon cancer, large bowel cancer,
uterine cancer, bile duct cancer, islet cell carcinoma,
adrenocortical cancer, bladder cancer, testis cancer, testicular
tumor, thyroid cancer, skin cancer, malignant carcinoid tumor,
malignant melanoma, osteosarcoma, soft tissue sarcoma,
neuroblastoma, Wilms tumor, retinoblastoma, melanoma, glioma and
the like, and of them, ovarian cancer, pancreatic cancer, stomach
cancer, gallbladder cancer, kidney cancer, prostatic cancer, breast
cancer, esophageal cancer, liver cancer, oral cavity cancer, colon
cancer, large bowel cancer, sarcoma, melanoma and glioma are
preferable, and particularly, ovarian cancer, pancreatic cancer,
stomach cancer and gallbladder cancer are preferable.
[0172] An agent containing the segment of a glycosylation-deficient
HGF of the present invention can be used in combination with other
anticancer agent and the like. As the other anticancer agent, there
are listed, for example, alkylating agents (for example, nitrogen
mustard, merphalan, cyclophosphamide, chlorambucil, thiotepa,
dibromomannitol, dibromodulcitol, carmustine, lomustine, semustine,
nimustine hydrochloride, chlorozotocine, ranimustine, busulphan,
procarbazine and the like), various antimetabolites (for example,
6-mercaptopurine, azathioprine, 6-thioguanine, thioinosine,
fluorouracil, tegafur, carmofur, doxifluridine, broxuridine,
cytarabine, enocitabine, methotrexate, trimethotrexate and the
like), antitumor antibiotics (for example, daunorubicin,
aclarubicin, doxorubicin, actinomycin D, mitomycin C, bleomycin,
peplomycin and the like), other antitumor agents (for example,
cisplatin, carboplatin, tamoxifen, L-asparaginase, aceglaton,
shizophyllan, picibanil, ubenimex, krestin and the like), antitumor
plant components (camptothecin, vindesine, vincristine, vinblastine
and the like), BRM (biological response modifier; for example,
tumor necrosis factor, indomethacin and the like), cell adhesion
inhibitors (for example, substance having RGD sequence, and the
like), matrix metalloproteinase inhibitors (for example,
marimastat, batimastat and the like), hormones (for example,
hydrocortisone, dexamethasone, methylprednisolone, prednisolone,
prasterone, betamethasone, triamcinolone, oxymetholone, nandrolone,
methenolone, fosfestrol, ethinyl estradiol, chlormadinone,
medroxyprogesterone and the like), vitamins (for example,
tocopherol and the like), antibiotics (for example, ampicillin,
sulbenicillin, cephalexin, cephaloridine, tobramycin,
chloramphenicol, tetracycline hydrochloride, kitasamycin,
lincomycin hydrochloride and the like) or chemotherapeutic agents
(for example, sulfamethizole, sulfamonomethoxine, enoxacin,
norfloxacin, ofloxacin, quinoxacin and the like), and the like.
Further, an agent containing the segment of a
glycosylation-deficient HGF of the present invention can also be
used in combination with surgical therapy and radiation therapy.
Therefore, a pharmaceutical composition containing the segment of a
glycosylation-deficient HGF of the present invention can be used
for suppressing invasion, suppressing growth, suppressing
metastasis, inducing apoptosis and/or inhibiting neovascularization
of tumor, in combination with other anticancer agents and the like
and/or surgical therapy or radiation therapy.
[0173] When an agent containing the segment of a
glycosylation-deficient HGF of the present invention is used as an
agent for preventing and treating diseases due to
neovascularization, there are exemplified diseases due to
neovascularization, such as rheumatic arthritis, psoriasis,
Osler-Webber syndrome, myocardial vasculogenesis, telangiectasis,
hemophilic arthritis, eye neovascularization diseases (for example,
diabetic retinopathy, retinophathy of prematurity, senile macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, perosis and the like), angiofibroma,
benign tumors (for example, angioma, acoustic neuroma,
neurofibroma, trachoma, pyogenic granuloma, and the like),
hematopoietic organ tumors including leukemia, solid tumors, tumor
metastasis, granulation of a wound and the like.
[0174] An agent containing the segment of a glycosylation-deficient
HGF of the present invention can be widely applied to prevention or
treatment of diseases caused by excessive or abnormal stimulation
of an endothelial cell. Such diseases are not particularly
restricted, and there are specifically exemplified intestinal
adhesion, Crohn's disease, atherosclerosis, scleroderma, excess
scar formation such as keloid and the like. Further, an agent
containing the segment of a glycosylation-deficient HGF of the
present invention, particularly, glycosylation-deficient NK4 is
useful as a birth control agent based on prevention of
neovascularization necessary for embryo implantation, and useful as
an agent for preventing or treating diseases associated with
neovascularization such as cat scratch disease and ulcer as a
pathologic result.
[0175] Further, the segment of a glycosylation-deficient HGF can be
used as an agent for treatment and prevention of infection diseases
such as Listeria and malaria.
[0176] A base sequence coding the segment of a
glycosylation-deficient HGF of the present invention can be used
also as a gene pharmaceutical composition. The target diseases in
gene therapy include the above-mentioned diseases.
EXAMPLES
[0177] The present invention will be further illustrated in detail
by the following examples, but the scope of the invention is not
limited to these examples.
[0178] Abbreviations used in examples are as described below.
[0179] HGF: hepatocyte growth factor
[0180] bFGF: basic fibroblast growth factor
[0181] LB: Luria-Bertani
[0182] Amp: ampicillin
[0183] SDS-PAGE: SDS-polyacrylamide gel electrophoresis
[0184] Tween 80: polyoxyethylene (20) sorbitan monooleate
Example 1
[0185] A base sequence coding 5 amino acid-deleted type NK4 shown
in SEQ ID NO: 3 was incorporated into a pCAGGS vector. The
resulting vector is designated pCAGGS-NK4.
[0186] For the purpose of introducing mutations into three
glycosylation sites (289th, 397th and 471st of SEQ ID NO: 2)
present in the NK4 protein, three mutagenic primers
(5'-phosphorylated) shown in Table 1 were synthesized and
site-directed mutagenesis was performed using the pCAGGS-NK4 vector
as a template. By this mutagenesis, Asn 289th and Asn 397th are
substituted by Gln, and Thr 471st is substituted by Gly, among
amino acid sequences of the 5 amino acid-deleted type NK4. Entire
amino acid sequence after incorporation of mutation is shown in SEQ
ID No:4. For the mutagenesis, QuikChange Multi Kit available from
STRATAGENE was utilized.
1TABLE 1 Sequence Base sequence number 5'-tgc gct gac aat act atg
caa gac SEQ ID NO: 5 act gat gtt cct ttg-3' 5'-ggc aaa aat tat atg
ggc cag tta SEQ ID NO: 6 tcc caa aca aga tct gg-3' 5'-tgc aaa cag
gtt ctc caa gtt tcc SEQ ID NO: 7 cag ctg gta tat gg-3'
[0187] The vector containing the introduced mutations was
transformed into a competent cell of E. coli XL10 Gold, and
Amp-resistant colonies were picked up on an LB/Amp plate. Plasmids
were extracted from the resulting each clone, and the intended
clone was screened by analyzing a base sequence on the coding
region of NK4. The vectors having intended three mutations in the
base sequence coding NK4 and no other mutation being confirmed were
selected and used in the subsequent experiments. The resultant
mutated vector is referred to as pCAGGS-NK4-NG.
[0188] Next, pCAGGS-NK4-NG was transfected into COS-7 cells. COS-7
cells were cultured in Dulbecco modified Eagle medium (DMEM)
supplemented with 10% fetal calf serum (FCS). The medium for the
cells was changed to serum-free DMEM just before transfection.
Transfection was conducted by a lipofection method using
LIPOFECTAMINE 2000 (Invitrogen). At 6 hours after transfection, the
medium was changed to DMEM containing 1% FCS and 5 mM benzamidine,
and in this procedure, heparin was added to a concentration of 1
.mu.g/mL. After 24 hours, the culture supernatant was collected
after filtration through a filter with a pore size of 0.22 .mu.m,
then, the medium was changed to a new medium (DMEM containing 1%
FCS, 1 .mu.g/mL heparin and 5 mM benzamidine), and culturing was
conducted further for 24 hours. After 24 hours, the culture
supernatant was again collected in the same manner. The collected
supernatant of the culture solution was preserved at -80.degree. C.
until purification thereof. The concentration of the
glycosylation-deficient NK4 secreted into the culture medium was
analyzed by ELISA.
[0189] The above-mentioned medium was thawed and mixed, and again
filtered through a filter with a pore size of 0.22 .mu.m, then,
added at a flow rate of 1 mL/min to HiTrap heparin (Bed volume: 5
mL)(Amersham Bioscience K.K.) equilibrated with 50 mM
Tris-hydrochloric acid (pH 7.5), 0.01% Tween 80 and 0.3 M sodium
chloride. The column was washed with 50 mM Tris-hydrochloric acid
(pH 7.5), 0.01% Tween 80 and 0.3 M sodium chloride, then, the NaCl
concentration was raised up to 2 M to elute glycosylation-deficient
NK4. Elution was conducted at a flow rate of 1 mL/min, and
fractionated at a rate of 2.5 mL/tube. A fraction containing the
glycosylation-deficient NK4 was collected, and the buffer was
exchanged to 50 mM Tris-hydrochloric acid (pH 7.5), 0.01% Tween 80
and 0.3 M sodium chloride by ultrafiltration. This was added at a
flow rate of 0.4 mL/min to a Mini S column (Bed volume: 0.8 mL)
(Amersham Bioscience K.K.) equilibrated with the same buffer. The
column was washed with 50 mM Tris-hydrochloric acid (pH 7.5), 0.01%
Tween 80 and 0.3 M sodium chloride, then, the NaCl concentration
was raised up to 1 M to elute glycosylation-deficient NK4. Elution
was conducted at a flow rate of 0.4 mL/min, and fractionated at a
rate of 0.4 mL/tube. A fraction containing the
glycosylation-deficient NK4 was collected, and the purification was
confirmed by SDS-PAGE.
[0190] An NK4 protein was also prepared using CHO cells according
to a method described in JP-A No. 2003-250549 (referred to as
glycosylated NK4).
[0191] The glycosylated NK4 and glycosylation-deficient NK4 were
reduced and allowed to migrate by SDS-PAGE, then, the gel was
stained with silver and the results are shown in FIG. 1. The band
of the glycosylated NK4 was confirmed at a position corresponding
to a molecular weight of 67 kDa. In the glycosylation-deficient
NK4, the band shifted to a position (molecular weight of 51 kDa of
NK4 protein backbone) corresponding to the molecular weight of
sugar chain deficiency.
Example 2
[0192] MDCK-3B cells were suspended in DMEM (containing 10% FCS)
and seeded on a 96-well plate to give a density of 5000 cells/well,
and HGF was added thereon to provide a concentration of 30 pM. In
this procedure, the glycosylated NK4 or glycosylation-deficient NK4
was added simultaneously to provide a concentration of 3 nM, 30 nM
or 85 nM, and cultured at 37.degree. C. for 20 hours, then, the
extent of scattering was observed by a microscope (FIG. 2). Though
the usual morphology of the MDCK-3B cell is as shown in FIG. 2A,
when HGF was added and cultured, cells scattered (FIG. 2B). When
the glycosylated NK4 was added simultaneously with HGF, scattering
of cells was suppressed, and the higher the concentration of the
glycosylated NK4 was, the more cell scattering was suppressed
(FIGS. 2C, D, E). The glycosylation-deficient NK4 suppressed
scattering of the MDCK-3B cell with the same degree as the
glycosylated NK4 (FIGS. 2G, H, I). That is, the
glycosylation-deficient NK4 showed an HGF antagonist activity of
the same degree as the glycosylated NK4. When HGF was not added,
the MDCK-3B cell showed no change even if the glycosylated NK4 or
glycosylation-deficient NK4 was added (FIGS. 2F, J).
Example 3
[0193] Human umbilical vein endothelial cells (HUVEC) were
suspended in MDCB131 medium (containing 5% FCS) and seeded on a
96-well plate at a density of 2000 cells/well, and cultured for 24
hours without adding bFGF. Then, the medium was changed to a new
MDCB131 medium (containing 5% FCS) and bFGF was simultaneously
added to give a concentration of 3 ng/mL, and cultured for 72
hours. In this procedure, the glycosylated NK4 or
glycosylation-deficient NK4 was added simultaneously with bFGF to
provide a concentration of 30 nM or 85 nM. The number of cells
(HUVEC) 72 hours after culturing was counted, and effects of the
glycosylated NK4 and glycosylation-deficient NK4 on suppressing
proliferation of HUVEC were compared (FIG. 3). When the
glycosylated NK4 or glycosylation-deficient NK4 was not added,
HUVEC proliferated by the action of bFGF, and the number of cells
thereof increased. When the glycosylated NK4 was added
simultaneously with bFGF, proliferation of HUVEC was suppressed,
and the higher the concentration of the glycosylated NK4 was, the
more proliferation of HUVEC was suppressed. The
glycosylation-deficient NK4 suppressed proliferation of HUVEC at
the same degree as the glycosylated NK4. That is, the
glycosylation-deficient NK4 had a neovascularization inhibitory
activity of the same degree as the glycosylated NK4.
Example 4
[0194] For examining the action of suppressing invasion of cancer
cells by the glycosylated NK4 and glycosylation-deficient NK4,
human gallbladder cancer cells (GB-dl) were suspended in DMEM
(containing 10% FCS), and added (15000 cells/chamber) into a
matrigel chamber (BD BIOCOAT Matrigel chamber, available from BD
Biosciences). The chamber was set on a 24-well plate, and DMEM
(containing 10% FCS) was added to the outside of the chamber. To
the DMEM medium outside the chamber, HGF was added to give a
concentration of 110 pM. In this procedure, the glycosylated NK4 or
glycosylation-deficient NK4 was added simultaneously to the DMEM
medium outside the chamber to provide a concentration of 1 nM, 11
nM or 85 nM and the cells were cultured at 37.degree. C. for 24
hours. Then, the number of GB-d1 cells invaded out of the chamber
was counted. The number of invading cells when only HGF was added
was defined as 100, and the number of cancer cells invaded out of
the chamber was shown as relative value (FIG. 4). The matrigel
imitates a basement membrane, and cancer cells in the chamber
invade out of the chamber by the action of HGF added outside of the
chamber. When the glycosylated NK4 or glycosylation-deficient NK4
was not added, a lot of cancer cells invaded out of the chamber by
the invasion-inducing action of HGF. When the glycosylated NK4 was
added simultaneously with HGF, invasion of cancer cells was
suppressed, and the higher the concentration of the glycosylated
NK4 was, the more invasion was suppressed. Further, the
glycosylation-deficient NK4 suppressed invasion of the GB-d1 cells
at the same degree as the glycosylated NK4.
Example 5
[0195] A DNA fragment coding mature polypeptide of
glycosylation-deficient NK4 (5-amino acid deletion type)
(represented from 32nd to 473rd in SEQ ID NO: 4) was prepared by
PCR using pCAGGS-NK4-NG described in Example 1 as a template, so
that the said sequence was flanked with sequence containing Not I
restriction site. The DNA fragment was digested with Not I and was
inserted into the Not I site of pPIC9K vector (Invitrogen) for
yeast expression, in which a foreign gene is driven by AOX1
promoter and the gene product is secreted from host cells using the
.alpha.-factor secretion signal. The constructed vector was
designated pPIC9K-NK4-NG. Then the vector was linearized with Sac I
digestion and was introduced by electroporation method into
spheroplasts of Pichia pastoris KM71 (Invitrogen), a methylotrophic
yeast. Transformants were selected on agar plates containing no
histidine. Because the KM71 strain has a mutation in the his4 gene
coding histidinol dehydrogenase responsible for histidine
synthesis, it can not grow without supplementation of histidine.
Therefore, only transformants to which pPIC9K vector containing
his4 gene was introduced can grow on plates without histidine. Two
hundred His+ transformants were picked up. Next,
Geneticin-resistant clones were selected from the His+
transformants. The pPIC9K vector contains the kanamycin gene that
confers resistance to Geneticin in Pichia. The level of Geneticin
resistance depends on the number of kanamycin genes integrated.
Therefore, transformants resistant to high level of Geneticin
contain multiple integrated copies of the vector, thereby showing
high expression of recombinant proteins.
[0196] For production of glycosylation-deficient NK4, one clone
(KM71-4-48) showing resistance to 4 mg/ml Geneticin was used. The
KM71-4-48 clone was grown in 5 ml of BMGY medium (1% yeast extract,
2% peptone, 100 mM potassium phosphate (pH 6), 1.34% yeast nitrogen
base, 0.00004% biotin, 1% glycerol) at 30.degree. C. with shaking
for 1 day, and cells were collected by centrifugation. Next, the
collected cells were inoculated to 1 ml of BMMY medium (1% yeast
extract, 2% peptone, 100 mM potassium phosphate (pH 6), 1.34% yeast
nitrogen base, 0.00004% biotin, 0.5% methanol) in order to induce
recombinant production, and culture was conducted at 30.degree. C.
with shaking for 5 days. Methanol was fed at a final concentration
of 0.5% at every 24 h. After 5 days, glycosylation-deficient NK4
secreted into the culture medium was analyzed. In Western blotting
using anti-human HGF polyclonal antibody, the band of
glycosylation-deficient NK4 in the culture medium was detected at a
size of 51 kDa corresponding to the lack of glycosylation (FIG. 5).
The concentration of glycosylation-deficient NK4 in the culture
medium was 2 .mu.g/ml on the basis of ELISA.
INDUSTRIAL APPLICABILITY
[0197] The segment of a glycosylation-deficient HGF of the present
invention is useful as an alternate for a segment of glycosylated
HGF.
Sequence CWU 1
1
7 1 494 PRT Homo sapiens alpha-chain of human hepatocyte growth
factor 1 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His
Val Leu 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala
Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys
Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu
Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala
Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys
Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe
Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly
His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120
125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His
Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp
Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly
Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu
Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu
Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240
His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245
250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys
Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile
Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro
Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr
Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys
Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr
Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys
Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365
Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370
375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr
Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys
Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile
Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys
Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr
Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg
Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp
His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg 485 490 2 489
PRT Homo sapiens alpha-chain of human hepatocyte growth factor of
five amino acid-deleted type 2 Met Trp Val Thr Lys Leu Leu Pro Ala
Leu Leu Leu Gln His Val Leu 5 10 15 Leu His Leu Leu Leu Leu Pro Ile
Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr
Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys
Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr
Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85
90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu
Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile
Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr
Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser
Ser Met Ile Pro His Glu His 145 150 155 160 Ser Tyr Arg Gly Lys Asp
Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg 165 170 175 Gly Glu Glu Gly
Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg 180 185 190 Tyr Glu
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 195 200 205
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 210
215 220 Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys
Phe 225 230 235 240 Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp
Asn Tyr Cys Arg 245 250 255 Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys
Tyr Thr Leu Asp Pro His 260 265 270 Thr Arg Trp Glu Tyr Cys Ala Ile
Lys Thr Cys Ala Asp Asn Thr Met 275 280 285 Asn Asp Thr Asp Val Pro
Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln 290 295 300 Gly Glu Gly Tyr
Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro 305 310 315 320 Cys
Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro 325 330
335 Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro
340 345 350 Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn
Ile Arg 355 360 365 Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met
Ser His Gly Gln 370 375 380 Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr
Met Gly Asn Leu Ser Gln 385 390 395 400 Thr Arg Ser Gly Leu Thr Cys
Ser Met Trp Asp Lys Asn Met Glu Asp 405 410 415 Leu His Arg His Ile
Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu 420 425 430 Asn Tyr Cys
Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr 435 440 445 Thr
Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys 450 455
460 Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile
465 470 475 480 Ser Cys Ala Lys Thr Lys Gln Leu Arg 485 3 1422 DNA
Homo sapiens NK4 of five amino acid-deleted type 3 atgtgggtga
ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60
ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat
120 gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact
gaagataaaa 180 accaaaaaag tgaatactgc agaccaatgt gctaatagat
gtactaggaa taaaggactt 240 ccattcactt gcaaggcttt tgtttttgat
aaagcaagaa aacaatgcct ctggttcccc 300 ttcaatagca tgtcaagtgg
agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360 aacaaagact
acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420
tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac
480 agctatcggg gtaaagacct acaggaaaac tactgtcgaa atcctcgagg
ggaagaaggg 540 ggaccctggt gtttcacaag caatccagag gtacgctacg
aagtctgtga cattcctcag 600 tgttcagaag ttgaatgcat gacctgcaat
ggggagagtt atcgaggtct catggatcat 660 acagaatcag gcaagatttg
tcagcgctgg gatcatcaga caccacaccg gcacaaattc 720 ttgcctgaaa
gatatcccga caagggcttt gatgataatt attgccgcaa tcccgatggc 780
cagccgaggc catggtgcta tactcttgac cctcacaccc gctgggagta ctgtgcaatt
840 aaaacatgcg ctgacaatac tatgaatgac actgatgttc ctttggaaac
aactgaatgc 900 atccaaggtc aaggagaagg ctacaggggc actgtcaata
ccatttggaa tggaattcca 960 tgtcagcgtt gggattctca gtatcctcac
gagcatgaca tgactcctga aaatttcaag 1020 tgcaaggacc tacgagaaaa
ttactgccga aatccagatg ggtctgaatc accctggtgt 1080 tttaccactg
atccaaacat ccgagttggc tactgctccc aaattccaaa ctgtgatatg 1140
tcacatggac aagattgtta tcgtgggaat ggcaaaaatt atatgggcaa cttatcccaa
1200 acaagatctg gactaacatg ttcaatgtgg gacaagaaca tggaagactt
acatcgtcat 1260 atcttctggg aaccagatgc aagtaagctg aatgagaatt
actgccgaaa tccagatgat 1320 gatgctcatg gaccctggtg ctacacggga
aatccactca ttccttggga ttattgccct 1380 atttctcgtt gtgaaggtga
taccacacct acaatagtct ga 1422 4 473 PRT Homo sapiens NK4 of five
amino acid-deleted type 4 Met Trp Val Thr Lys Leu Leu Pro Ala Leu
Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile
Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr
Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys
Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr
Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85
90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu
Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile
Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr
Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser
Ser Met Ile Pro His Glu His 145 150 155 160 Ser Tyr Arg Gly Lys Asp
Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg 165 170 175 Gly Glu Glu Gly
Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg 180 185 190 Tyr Glu
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 195 200 205
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 210
215 220 Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys
Phe 225 230 235 240 Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp
Asn Tyr Cys Arg 245 250 255 Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys
Tyr Thr Leu Asp Pro His 260 265 270 Thr Arg Trp Glu Tyr Cys Ala Ile
Lys Thr Cys Ala Asp Asn Thr Met 275 280 285 Gln Asp Thr Asp Val Pro
Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln 290 295 300 Gly Glu Gly Tyr
Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro 305 310 315 320 Cys
Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro 325 330
335 Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro
340 345 350 Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn
Ile Arg 355 360 365 Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met
Ser His Gly Gln 370 375 380 Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr
Met Gly Gln Leu Ser Gln 385 390 395 400 Thr Arg Ser Gly Leu Thr Cys
Ser Met Trp Asp Lys Asn Met Glu Asp 405 410 415 Leu His Arg His Ile
Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu 420 425 430 Asn Tyr Cys
Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr 435 440 445 Thr
Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys 450 455
460 Glu Gly Asp Thr Thr Pro Gly Ile Val 465 470 5 39 DNA Artificial
Sequence Synthetic construct 5 tgcgctgaca atactatgca agacactgat
gttcctttg 39 6 41 DNA Artificial Sequence Synthetic construct 6
ggcaaaaatt atatgggcca gttatcccaa acaagatctg g 41 7 38 DNA
Artificial Sequence Synthetic construct 7 tgcaaacagg ttctccaagt
ttcccagctg gtatatgg 38
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