U.S. patent application number 11/394422 was filed with the patent office on 2007-01-11 for monitoring and modulating hgf/hgfr activity.
Invention is credited to Michael Detmar, Kentaro Kajiya.
Application Number | 20070010443 11/394422 |
Document ID | / |
Family ID | 36638370 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010443 |
Kind Code |
A1 |
Detmar; Michael ; et
al. |
January 11, 2007 |
Monitoring and modulating HGF/HGFR activity
Abstract
Provided are methods and compositions for the modulation of
hepatocyte growth factor activity to regulate lymphatic vessel
development and function. Methods and composition for the
monitoring and treatment of skin disorders, lymphedema, and
metastatic cancers are disclosed. Also described are methods of
identifying inhibitors of hepatocyte growth factor dependent
lymphangiogenesis.
Inventors: |
Detmar; Michael; (Boppelsen,
CH) ; Kajiya; Kentaro; (Yokohama, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
36638370 |
Appl. No.: |
11/394422 |
Filed: |
March 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60667463 |
Mar 31, 2005 |
|
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|
Current U.S.
Class: |
514/9.5 ;
435/7.2; 514/13.3; 514/18.7; 514/19.3; 514/44R |
Current CPC
Class: |
A61P 17/02 20180101;
A61P 43/00 20180101; A61P 35/02 20180101; A61P 7/10 20180101; G01N
2500/10 20130101; A61P 17/06 20180101; A61K 38/1833 20130101; G01N
33/5064 20130101; A61P 35/00 20180101; C07K 16/22 20130101; A61P
17/00 20180101; G01N 2333/4753 20130101; G01N 2500/04 20130101;
A61P 17/16 20180101; A61K 2039/505 20130101; A61P 35/04 20180101;
C07K 14/4753 20130101 |
Class at
Publication: |
514/012 ;
514/044; 435/007.2 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 48/00 20060101 A61K048/00; G01N 33/567 20060101
G01N033/567 |
Claims
1. A method of treating a subject having an unwanted skin condition
comprising increasing hepatocyte growth factor activity to thereby
treat the disorder.
2. The method of claim 1, wherein the unwanted skin condition is a
condition that affects the structure of the skin.
3. The method of claim 1, wherein the condition can be caused by a
genetic factor.
4. The method of claim 3, wherein the genetic factor is
epidermolysis.
5. The method of claim 1, wherein the condition is caused by an
environmental factor.
6. The method of claim 5, wherein the environmental factor is
ultraviolet radiation.
7. The method of claim 1, wherein the unwanted condition is aged
skin.
8. The method of claim 1, wherein the unwanted condition is
psoriasis.
9. The method of claim 1, wherein the unwanted condition is rosacea
dermatosis.
10. The method of claim 1, wherein the unwanted condition is skin
damage caused by photoradiation.
11. The method of claim 1, wherein increasing hepatocyte growth
factor activity comprises administering to the subject an agonist
of hepatocyte growth factor activity.
12. The method of claim 11, wherein the agonist of hepatocyte
growth factor activity is selected from the group consisting of: a
hepatocyte growth factor polypeptide or a biologically active
fragment or analog thereof, a nucleic acid encoding a hepatocyte
growth factor or a biologically active fragment or analog thereof,
and an agonist of hepatocyte growth factor.
13. The method of claim 11, wherein the agonist of hepatocyte
growth factor activity is an agent effective to increase the
endogenous level of hepatocyte growth factor.
14. A method of treating a subject having lymphedema comprising
increasing hepatocyte growth factor activity to thereby treat the
lymphedema.
15. The method of claim 14, wherein increasing hepatocyte growth
factor activity comprises administering to the subject an agonist
of hepatocyte growth factor activity.
16. The method of claim 15, wherein the agonist of hepatocyte
growth factor activity is selected from the group consisting of: a
hepatocyte growth factor polypeptide or a biologically active
fragment or analog thereof, a nucleic acid encoding a hepatocyte
growth factor or a biologically active fragment or analog thereof,
and an agonist of hepatocyte growth factor.
17. The method of claim 15, wherein the agonist of hepatocyte
growth factor activity is an agent effective to increase the
endogenous level of hepatocyte growth factor.
18. A method of treating a subject having or at risk for a
neoplastic disorder comprising inhibiting hepatocyte growth factor
activity to thereby treat or reduce the risk of the neoplastic
disorder.
19. The method of claim 18, wherein the neoplastic disorder is a
cancer.
20. The method of claim 19, wherein the cancer is characterized by
a risk of metastasis to a lymph node.
21. The method of claim 18, wherein inhibiting hepatocyte growth
factor activity comprises administering to the subject an
antagonist of hepatocyte growth factor activity.
22. The method of claim 21, wherein the antagonist of hepatocyte
growth factor activity is selected from the group consisting of: a
hepatocyte growth factor nucleic acid molecule that can bind to
cellular hepatocyte growth factor mRNA and inhibit expression of
the protein, an antibody that specifically binds to a hepatocyte
growth factor protein, a soluble hepatocyte growth factor receptor,
a dominant negative hepatocyte growth factor protein or fragment
thereof, and an agent that decreases hepatocyte growth factor
nucleic acid expression.
23. The method of claim 21, wherein the antagonist of hepatocyte
growth factor activity is an agent effective to decrease the
endogenous level of hepatocyte growth factor.
24. A method of treating a subject having or at risk for a
neoplastic disorder comprising inhibiting .alpha.9 integrin
activity to thereby treat or reduce the risk of the neoplastic
disorder.
25. The method of claim 24, wherein inhibiting .alpha.9 integrin
activity comprises administering to the subject an antagonist of
.alpha.9 integrin activity.
26. The method of claim 25, wherein the antagonist of .alpha.9
integrin activity is selected from the group consisting of: an
.alpha.9 integrin nucleic acid molecule that can bind to cellular
.alpha.9 integrin mRNA and inhibit expression of the protein, an
antibody that specifically binds to an .alpha.9 integrin, a soluble
.alpha.9 integrin receptor, a dominant negative .alpha.9 integrin
protein or fragment thereof, and an agent that decreases .alpha.9
integrin nucleic acid expression.
27. A method of identifying a compound that inhibits hepatocyte
growth factor-dependent lymphatic endothelial cell proliferation or
migration, comprising: providing a lymphatic endothelial cell
expressing the hepatocyte growth factor receptor; contacting the
lymphatic endothelial cell with hepatocyte growth factor and a test
compound; and determining whether proliferation of the lymphatic
endothelial cell is decreased in the presence of the test compound,
a decrease in the proliferation being an indication that the test
compound inhibits hepatocyte growth factor-dependent lymphatic
endothelial cell proliferation.
28. The method of claim 27, wherein the lymphatic endothelial cell
is a mammalian cell selected from the group consisting of: mouse,
rat, rabbit, hamster, and human.
29. The method of claim 28, wherein the mammalian cell expresses
Prox1 and the hyaluronan receptor LYVE-1.
30. The method of claim 27, wherein the test compound is a peptide
or an antibody.
31. The method of claim 27, wherein the lymphatic endothelial cell
expresses a recombinant hepatocyte growth factor receptor or a
mutant thereof.
32. A method of identifying a compound that inhibits hepatocyte
growth factor-dependent lymphangiogenesis comprising: providing
lymphatic endothelial cells expressing the hepatocyte growth factor
receptor; contacting the lymphatic endothelial cells with
hepatocyte growth factor and a test compound; and determining if
lymphangiogenesis is decreased, wherein a decrease in
lymphangiogenesis is an indication that the compound inhibits
hepatocyte growth factor-dependent lymphangiogenesis.
33. The method of claim 32, wherein the lymphatic endothelial cells
are mammalian cells selected from the group consisting of: mouse,
rat, rabbit, hamster, and human.
34. The method of claim 33, wherein the mammalians cell express
Prox1 and the hyaluronan receptor LYVE-1.
35. The method of claim 32, wherein the test compound is a peptide
or an antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application Ser.
No. 60/667,463, filed on Mar. 31, 2005. The contents of this
application are hereby incorporated by reference in their
entirety.
BACKGROUND
[0002] Hepatocyte growth factor (HGF) is a growth factor that can
be found in human serum. The HGF receptor (HGFR) has been
identified as the product of the c-Met proto-oncogene. See, e.g.,
Bottaro et al., Science, 251:802-804 (1991); Naldini et al.,
Oncogene, 6:501-504 (1991); WO 92/13097; and WO 93/15754.
SUMMARY
[0003] In one aspect, the disclosure features a method of treating
a subject having an unwanted skin condition, e.g., a condition that
impairs skin structure. The method includes administering, to the
subject, a therapeutically effective amount of an HGF/HGFR
modulator. In one aspect, the disclosure features the use of a
therapeutically effective amount of an HGF/HGFR modulator (e.g., an
agonist or antagonist) in the preparation of a medicament.
[0004] In one embodiment the modulator is an HGF/HGFR agonist. An
HGF/HGFR agonist is an agent that directly or indirectly increases
HGF/HGFR activity in the subject.
[0005] The agonist can be used to treat a condition or in the
preparation of a medicament for the treatment of a condition in
which increased lymphatic vessel formation is desired. Such
conditions included lymphedema, e.g., acquired lymphedema. Examples
of acquired lymphedema include lymphedema acquired after surgery or
radiation therapy or lymphedema caused at least in part by an
infection, e.g., by a pathogen, e.g., an infection such as
filariasis. Other conditions in which increased lymphatic vessel
formation is desired include aged skin or damaged skin, e.g.,
UVB-damaged skin. Still other conditions include those caused in
part by a genetic or environmental factor, e.g., ultraviolet
radiation. For example, the condition is epidermolysis, e.g.,
epidermolysis caused by aging or excessive exposure to ultra-violet
light.
[0006] Many HGF/HGFR agonists increase HGFR signalling activity.
Examples of HGF/HGFR agonists include a protein that includes a
hepatocyte growth factor polypeptide (e.g., as modified into its
mature heterodimeric form) or a biologically active fragment or
analog thereof, a nucleic acid encoding a hepatocyte growth factor
or a biologically active fragment or analog thereof. Other proteins
and molecules, e.g., antibodies and small molecules, can also be
used increase HGFR activity. For example, antibodies that bind, and
optionally crosslink (e.g., dimerize) HGFR can be used to HGFR
activity.
[0007] For example, the unwanted condition is an inflammatory or
autoimmune skin disorder (e.g., psoriasis), or rosacea dermatosis.
A therapeutically effective amount of an HGF/HGFR agonist can be
used in the preparation of a medicament for the treatment of such
an inflammatory or autoimmune skin disorder or rosacea
dermatosis.
[0008] In one embodiment, the modulator is an HGF/HGFR antagonist.
An HGF/HGFR antagonist is an agent that directly or indirectly
decreases HGF/HGFR activity in a cell or in the subject.
Antagonists include nucleic acids and proteins, e.g., antibodies or
soluble HGF receptor fragments. For example, the antagonist can be
a protein that interacts with HGF and, e.g., reduces HGF binding
affinity to cell surface HGFR. For example, the protein can be (i)
an antibody that recognizes HGF or HGFR, or (ii) a protein that
includes a extracellular region of the HGFR, e.g., a soluble HGF
receptor (e.g., fused to an Fc domain). Examples of antagonists
include: a nucleic acid molecule that can bind or otherwise inhibit
HGF mRNA, e.g., mRNA production, processing, or translation. Still
other antagonists include: a dominant negative HGF protein or
fragment thereof and an agent which decreases HGF nucleic acid
expression (e.g., an artificial transcription factor or nucleic
acid encoding an artificial transcription factor).
[0009] In some implementations, the modulator decreases the
endogenous level of HGF or HGFR.
[0010] In one aspect, the disclosure features a method of treating
a subject who has or is at risk for a neoplastic disorder, e.g., a
metastatic cancer, particularly one that includes cell metastasis
within lymph vessels or a cancer that has the potential to
metastasize to lymph nodes. The method includes administering, to
the subject, a therapeutically effective amount of an HGF/HGFR
antagonist that decreases HGF/HGFR activity in the subject. A
therapeutically effective amount of an HGF/HGFR antagonist can be
used in the preparation of a medicament for the treatment of a
subject who has or is at risk for a neoplastic disorder, e.g., a
metastatic cancer.
[0011] Antagonists include nucleic acids and proteins, e.g.,
antibodies or soluble HGF receptor fragments. For example, the
antagonist can be a protein or other agent that interacts with HGF
and, e.g., reduces HGF binding affinity to cell surface HGFR. For
example, the protein can be an antibody HGF or a extracellular
region of the HGFR, e.g., a soluble HGF receptor. Examples of
antagonists include: a nucleic acid molecule that can bind or
otherwise inhibit HGF mRNA, e.g., mRNA production, processing, or
translation. Still other antagonists include: a dominant negative
HGF protein or fragment thereof and an agent which decreases HGF
nucleic acid expression (e.g., an artificial transcription factor
or nucleic acid encoding an artificial transcription factor).
[0012] In another aspect, the disclosure features a method of
treating a subject who has or is at risk for a neoplastic disorder,
e.g., a metastatic cancer, particularly one that includes cell
metastasis within lymph vessels or a cancer that has the potential
to metastasize to lymph nodes. The method includes administering,
to the subject, a therapeutically effective amount of an .alpha.9
integrin antagonist that decreases .alpha.9 integrin activity in
the subject. For example, the antagonist is a protein or other
agent that interacts with .alpha.9 integrin or a counterpart
integrin beta subunit and, e.g., reduces integrin binding affinity
to cells. For example, the protein can be an antibody to .alpha.9
integrin or a counterpart integrin beta subunit or a extracellular
region of an .alpha.9 integrin receptor. A therapeutically
effective amount of an .alpha.9 integrin antagonist can be used in
the preparation of a medicament for the treatment of a subject who
has or is at risk for a neoplastic disorder, e.g., a metastatic
cancer.
[0013] Examples of antagonists include: a nucleic acid molecule
that can bind or otherwise inhibit .alpha.9 integrin mRNA, e.g.,
mRNA production, processing, or translation. Still other
antagonists include: a dominant negative .alpha.9 integrin protein
or fragment thereof and an agent which decreases .alpha.9 integrin
nucleic acid expression (e.g., an artificial transcription factor
or nucleic acid encoding an artificial transcription factor).
[0014] In still another aspect, the disclosure features a method of
evaluating a cell or a subject (e.g., using cells obtained from the
subject). The method includes evaluating integrin .alpha.9 and
stanniocalcin 1 mRNA or protein expression in the cell or in cells
from the subject. The method can be used to evaluate HGF activity
in the subject. For example, the cell or cells obtained from the
subject include endothelial cells, e.g., lymph endothelial cells
(LEC). The cell or subject can be treated, e.g., before, during, or
after the evaluating, with an agent described herein, e.g., an
agonist or antagonist of HGF/HGFR. The method can be used to
monitor a subject who has or is at risk for a disorder described
herein and who may be treated with an agent described herein.
[0015] In another aspect, the disclosure features a method of
identifying a compound that modulates lymphatic endothelial cell
activity, e.g., proliferation or migration. The method includes:
providing a cell or organism in which HGF/HGFR activity can be
monitored; contacting the cell or organism with a test compound;
and evaluating HGF/HGFR activity in the cell or organism. For
example, the cell includes a reporter of HGF/HGFR activity or an
organism that comprises such a cell. HGF/HGFR activity can be
evaluated by assaying, e.g., protein or mRNA expression or reporter
activity. A change in reporter activity or other relevant
parameter, for example, indicates a change in HGF/HGFR activity.
The method can further include evaluating the effect of the test
compound on cell proliferation or cell migration, e.g., lymphatic
endothelial cell proliferation or migration.
[0016] In one embodiment, the reporter is a gene that comprises a
sequence encoding a detectable protein and an operably linked
promoter that includes a region of the promoter of the HGF or HGF-R
gene, e.g. region from the transcription start site to a position
at least 100, 200, 300, 500, 800, 1000, 2000, or 5000 bases
upstream, from the initiator MET codon to a position at least 100,
200, 300, 500, 800, 1000, 2000, or 5000 bases upstream, or from the
TATA box to a position at least 100, 200, 300, 500, 800, 1000,
2000, or 5000 bases upstream.
[0017] The cell or organism is generally mammalian, e.g., human or
non-human, e.g., mouse, rat, hamster, guinea pig, monkey and so
forth.
[0018] In another aspect, the disclosure features a method of
identifying a compound that modulates endothelial cell activity,
for example, a compound that inhibits hepatocyte growth
factor-dependent lymphatic endothelial cell proliferation or
migration. The method includes: providing an endothelial cell
(e.g., a lymphatic endothelial cell) expressing a hepatocyte growth
factor receptor; contacting the endothelial cell with hepatocyte
growth factor and a test compound; and evaluating the cell, e.g.,
for a property, e.g., a property regulated by HGF/HGFR, such as
proliferation or migration. For example, the method can include
determining whether proliferation or migration of the lymphatic
endothelial cell is altered in the presence of the test compound. A
decrease in proliferation or migration can indicate that the test
compound inhibits hepatocyte growth factor-dependent lymphatic
endothelial cell proliferation.
[0019] The endothelial cell can be a mammalian cell, e.g., a mouse,
rat, rabbit, hamster, or human cell. The cell can be cultured or
isolated; for example, the cell is from a cell line or a primary
cell. In one embodiment, the cell expresses Prox1 and the
hyaluronan receptor LYVE-1.
[0020] The method can include evaluating the test compound in the
presence of another HGF/HGFR pathway modulator, e.g., in the
presence of a protein that includes soluble HGF, a protein that
includes a soluble extracellular domain of HGFR, or antibody to HGF
or HGFR.
[0021] The method can include evaluating tyrosine phosphorylation
of the hepatocyte growth factor receptor, e.g., to determine if the
test compound causes a decrease. In one embodiment, the lymphatic
endothelial cell expresses a recombinant hepatocyte growth factor
receptor or a mutant thereof.
[0022] The method can further include administering the test
compound to an organism, e.g., a human or non-human mammal. The
method can further include formulating a test compound or a
modified test compound that retains the biological activity of the
test compound as a pharmaceutical composition, e.g., by combining
the compound with a pharmaceutically acceptable carrier.
[0023] In still another aspect, the disclosure features a method
for evaluating a test compound, e.g., a compound that is topically
applied to a test organism, e.g., a transgenic organism that
includes a reporter of HGF/HGFR pathway activity. The method
includes contacting a test compound to the test organism and
evaluating HGF/HGFR pathway activity. For example, the evaluating
can include evaluating protein or mRNA expression of HGF, HGFR, or
a gene or gene product that is regulated by HGFR, e.g., .alpha.9
integrin or stanniocalcin 1. The method can also include evaluating
the test compound in the presence of another HGF/HGFR pathway
modulator, e.g., in the presence of a protein that includes soluble
HGF, a protein that includes a soluble extracellular domain of
HGFR, or antibody to HGF or HGFR.
[0024] In one embodiment, the reporter is a gene that includes a
sequence encoding a detectable protein and an operably linked
promoter that includes a region of the promoter of the HGF, HGFR,
.alpha.9 integrin or stanniocalcin 1 gene, e.g. region from the
transcription start site to a position at least 100, 200, 300, 500,
800, 1000, 2000, or 5000 bases upstream, from the initiator MET
codon to a position at least 100, 200, 300, 500, 800, 1000, 2000,
or 5000 bases upstream, or from the TATA box to a position at least
100, 200, 300, 500, 800, 1000, 2000, or 5000 bases upstream. The
method can also include evaluating two or more such reporters.
[0025] The method can include selecting a test compound (e.g., from
a library of test compounds), if it increases or decreases HGF/HGFR
pathway activity. A selected test compound can formulated, e.g., a
pharmaceutical composition, e.g., suitable for topical
administration or other route of administration. The method can
further include administering the pharmaceutical composition to a
subject, e.g., a subject having or at risk for a disorder described
herein.
[0026] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the claims.
All cited patents, applications, and references are incorporated
herein by reference.
DESCRIPTION OF DRAWINGS
[0027] FIG. 1 shows quantitative RT-PCR data for lymphatic cell
lineage marker mRNA levels and quantitative RT-PCR and immunoblot
assays for HGFR mRNA and protein, respectively.
[0028] FIG. 2 shows dual immunofluorescence staining for the
lymphatic marker LYVE-1 (green) and for HGFR (red), in lymphatic
vessels.
[0029] FIG. 3 shows dual immunofluorescence for the lymphatic
marker LYVE-1 (green) and HGFR (red) in endothelial cells of lymph
sacs during mouse embryonic development.
[0030] FIG. 4 shows data for an lymphatic endothelial cells (LEC)
proliferation and migration assay for LEC cells treated with
HGF.
[0031] FIG. 5 shows data for LEC tube formation in vitro, in
response to HGF and lymphatic vessel formation in vivo.
[0032] FIG. 6 shows dual immunofluorescence staining of lymphatic
vessels for LYVE-1 (green) and CD31 (red) after experimental skin
inflammation.
[0033] FIG. 7 shows QPCR data for integrin .alpha.9 and
stanniocalcin 1 mRNA levels in LEC after treatment with HGF.
[0034] FIG. 8 shows representative histologic images (H&E stain
and immunofluorescence to detect podoplanin) of the ileum in HGF
transgenic (FIGS. 8 B,D,F,H) and wild-type mice (FIGS. 8 A,C,E,G).
Scale bars: 100 .mu.m.
[0035] FIG. 9 shows double immunofluorescence analyses of mouse ear
sections for LYVE-1 (green) and CD31 (red). Induction of
LYVE-1-positive lymphatic vessel formation was observed at 14 days
after implantation of HGF-containing slow-release pellets (9B and
C; arrowheads) but not of control pellets (9A). Systemic treatment
with a blocking anti-VEGFR-3 antibody (9C) did not inhibit
HGF-induced lymphatic vessel formation, as compared with control
IgG treatment (9B). Newly formed blood vessels were observed in all
samples. P: Pellet. Scale bars: 100 .mu.m.
DETAILED DESCRIPTION
[0036] We have found that, among other things, the hepatocyte
growth factor receptor (HGFR) is highly expressed in lymphatic
endothelial cells (LEC) of the lymphatic system. Treatment of LEC
with HGF promotes proliferation, migration, and organization of
LECs into vessels. In addition, administration of HGF to mice
potently promoted new lymphatic vessel formation. Furthermore,
induction of LEC migration was largely mediated via .alpha.9
integrin.
[0037] Accordingly, it is possible to treat pathologies affected by
the lymphatic system by modulating HGF/HGFR activity. A subject can
also be diagnosed by evaluating a parameter that assesses HGF/HGFR
activity. Agents that modulate HGF/HGFR activity can be identified,
e.g., using the assay and screening methods described herein.
I. HGF and HGFR
[0038] The mature form of human HGF (hHGF), corresponding to the
major form purified from human serum, is a disulfide linked
heterodimer derived by proteolytic cleavage of the human
pro-hormone between amino acids R494 and V495. This cleavage
process generates a molecule composed of an .alpha. subunit of 440
amino acids (M.W. 69 kDa) and a .beta. subunit of 234 amino acids
(M.W. 34 kDa). The .alpha. and the .beta. chains are produced from
a single open reading frame coding for a pre-pro precursor protein.
In the predicted primary structure of an exemplary mature hHGF, an
interchain S-S bridge is formed between Cys 487 of the .alpha.
chain and Cys 604 in the .beta. chain (see, e.g., Nakamura et al.,
Nature 342:440-443, (1989)). The N-terminus of the .alpha. chain is
preceded by 54 amino acids, starting with a methionine group. This
segment includes a characteristic hydrophobic leader (signal)
sequence of 31 residues and the prosequence. The .alpha. chain
starts at amino acid (aa) 55, and contains four Kringle domains.
The Kringle 1 domain extends from about aa 128 to about aa 206, the
Kringle 2 domain is between about aa 211 and about aa 288, the
Kringle 3 domain is defined as extending from about aa 303 to about
aa 383, and the Kringle 4 domain extends from about aa 391 to about
aa 464 of the .alpha. chain. An exemplary HGF contains four
putative glycosylation sites, which are located at positions 294
and 402 of the .alpha.-chain and at positions 566 and 653 of the
.beta.-chain.
[0039] The HGF receptor (HGFR) has been identified as the product
of the c-Met proto-oncogene, Bottaro et al., Science, 251:802-804
(1991); Naldini et al., Oncogene, 6:501-504 (1991); WO 92/13097
published Aug. 6, 1992; WO 93/15754 published Aug. 19, 1993. The
receptor (AKA c-Met) typically comprises, in its native form, a
190-kDa heterodimeric (a disulfide-linked 50-kDa .alpha.-chain and
a 145-kDa .beta.-chain) membrane-spanning tyrosine kinase protein
(Park et al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987)).
[0040] The binding activity of HGF to its receptor is believed to
be conveyed by a functional domain located in the N-terminal
portion of the HGF molecule, including the first two Kringle
domains (Matsumoto et al., Biochem. Biophys. Res. Commun.,
181:691-699 (1991); Hartmann et al. Proc. Natl. Acad. Sci.,
89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510 (1992);
Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1991)). Upon
HGF binding, the c-Met protein becomes phosphorylated on tyrosine
residues of the 145-kDa .beta.-subunit.
[0041] With respect to the methods disclosed herein, HGF can be
produced as a purified polypeptide, for which the amino acid
sequence can be at least 80% identical (i.e., 85%, 87%, 89%, 90%,
92%, 94%, 96%, 98%, 99%, or 100% identical) to any of SEQ ID
NOs:1-4 listed below or other naturally occurring variants of HGF.
The receptor for HGF (HGFR) can be produced as a purified
polypeptide, for which the amino acid sequence can be at least 80%
identical (i.e., 85%, 87%, 89%, 90%, 92%, 94%, 96%, 98%, 99%, or
100% identical) to any of SEQ ID NOs:5-8 listed below or other
naturally occurring variants of HGFR. TABLE-US-00001 Human HGF (SEQ
ID NO:1) 1 MWVTKLLPAL LLQHVLLHLL LLPIAIPYAE GQRKRRNTIH EFKKSAKTTL
51 IKIDPALKIK TKKVNTADQC ANRCTRNKGL PFTCKAFVFD KARKQCLWFP 101
FNSMSSGVKK EFGHEFDLYE NKDYIRNCII GKGRSYKGTV SITKSGIKCQ 151
PWSSMIPHEH SFLPSSYRGK DLQENYCRNP RGEEGGPWCF TSNPEVRYEV 201
CDIPQCSEVE CMTCNGESYR GLMDHTESGK ICQRWDHQTP HRHKFLPERY 251
PDKGFDDNYC RNPDGQPRPW CYTLDPHTRW EYCAIKTCAD NTMNDTDVPL 301
ETTECIQGQG EGYRGTVNTI WNGIPCQRWD SQYPHEHDMT PENFKCKDLR 351
ENYCRNPDGS ESPWCFTTDP NIRVGYCSQI PNCDMSHGQD CYRGNGKNYM 401
GNLSQTRSGL TCSMWDKNME DLHRHIFWEP DASKLNENYC RNPDDDAHGP 451
WCYTGNPLIP WDYCPISRCE GDTTPTIVNL DHPVISCAKT KQLRVVNGIP 501
TRTNIGWMVS LRYRNKHICG GSLIKESWVL TARQCFPSRD LKDYEAWLGI 551
HDVHGRGDEK CKQVLNVSQL VYGPEGSDLV LMKLARPAVL DDFVSTIDLP 601
NYGCTIPEKT SCSVYGWGYT GLINYDGLLR VAHLYIMGNE KCSQHHRGKV 651
TLNESEICAG AEKIGSGPCE GDYGGPLVCE QHKMRMVLGV IVPGRGCAIP 701
NRPGIFVRVA YYAKWIHKII LTYKVPQS Chimpanzee HGF (SEQ ID NO:2) 1
MWVTKLLPAL LLQHVLLHLL LLPIAIPYAE GQRKRRNTIH EFKKSAKTTL 51
IKIDPALKIK TKKVNTADQC ANRCTRNKGL PFTCKAFVFD KARKQCLWFP 101
FNSMSSGVKK EFGHEFDLYE NKGHETFGRF LPSSYRGKDL QENYCRNPRG 151
EEGGPWCFTS NPEVRYEVCD IPQCSEVECM TCNGESYRGL MDHTESGKIC 201
QRWDHQTPHR HKFLPERYPD KGFDDNYCRN PDGQPRPWCY TLDPHTRWEY 251
CAIKTCADNT MNDTDVPLET TECIQGQGEG YRGTVNTIWN GIPCQRWDSQ 301
YPHEHDMTPE NFKCKDLREN YCRNPDGSES PWCFTTDPNI RVGYCSQIPN 351
CDMSHGQDCY RGNGKNYMGN LSQTRSGLTC SMWDKNMEDL HRHIFWEPDA 401
SKLNENYCRN PDDDAHGPWC YTGNPLIPWD YCPISRCEGD TTPTIVNLDH 451
PVISCAKTKQ LRVVNGIPTR TNVGWMVSLR YRNKHICGGS LIKESWVLTA 501
RQCFPSRDLK DYEAWLGIHD VHGRGDEKCK QVLNVSQLVY GPEGSDLVLM 551
KLARPAVLDD FVSTIDLPNY GCTIPEKTSC SVYGWGYTGL INYDGLLRVA 601
HLYIMGNEKC SQHHRGKVTL NESEICAGAE KIGSGPCEGD YGGPLVCEQH 651
KMRMVLGVIV PGRGCAIPNR PGIFVRVAYY AKWIHKIILT YKVPQS Mouse HGF (SEQ
ID NO:3) 1 MMWGTKLLPV LLLQHVLLHL LLLHVAIPYA EGQKKRRNTL HEFKKSAKTT
51 LTKEDPLLKI KTKKVNSADE CANRCIRNRG FTFTCKAFVF DKSRKRCYWY 101
PFNSMSSGVK KGFGHEFDLY ENKDYIRNCI IGKGGSYKGT VSITKSGIKC 151
QPWNSMIPHE HSFLPSSYRG KDLQENYCRN PRGEEGGPWC FTSNPEVRYE 201
VCDIPQCSEV ECMTCNGESY RGPMDHTESG KTCQRWDQQT PHRHKFLPER 251
YPDKGFDDNY CRNPDGKPRP WCYTLDPDTP WEYCAIKTCA HSAVNETDVP 301
METTECIQGQ GEGYRGTSNT IWNGIPCQRW DSQYPHKHDI TPENFKCKDL 351
RENYCRNPDG AESPWCFTTD PNIRVGYCSQ IPKCDVSSGQ DCYRGNGKNY 401
MGNLSKTRSG LTCSMWDKKM EDLHRHIFWE PDASKLNKNY CRNPDDDAHG 451
PWCYTGNPLI PWDYCPISRC EGDTTPTIVN LDHPVISCAK TKQLRVVNGI 501
PTQTTVGWMV SLKYRNKHIC GGSLIKESWV LTARQCFPAR NKDLKDYEAW 551
LGIHDVHERG EEKRKQILNI SQLVYGPEGS DLVLLKLARP AILDNFVSTI 601
DLPSYGCTIP EKTTCSTYGW GYTGLINADG LLRVAHLYIM GNEKCSQHHQ 651
GKVTLNESEL CAGAEKIGSG PCEGDYGGPL ICEQHKMRMV LGVIVPGRGC 701
AIPNRPGIFV RVAYYAKWIH KVILTYKL Rat HGF (SEQ ID NO:4) 1 MWVTKLLPAL
LLQHVLLHLL LLPIAIPYAE GHKKRRNTIH EFKKSAKTTL 51 IKIDPALKIK
TKKVNTADQC ANRCTRNNGL PFTCKAFVFD KARKQCLWFP 101 FNSMSSGVKK
EFGHEFDLYE NKDYIRNCII GKGRSYKGTV SITKSGIKCQ 151 PWSSMIPHEH
SFLPSSYRGK DLQENYCRNP RGEEGGPWCF TSNPEVRYEV 201 CDIPQCSEVE
CMTCNGESYR GLMDHTESGK ICQRWDHQTP HRHKFLPERY 251 PDKGFDDNYC
RNPDGQPRPW CYTLDPHTRW EYCAIKTCAD NTVNDTDVPM 301 ETTECIQGQG
EGYRGTANTI WNGIPCQRWD SQYPHKHDMT PENFKCKDLR 351 ENYCRNPDGS
ESPWCFTTDP NIRVGYCSQI PNCDMSNGQD CYRGNGKNYM 401 GNLSQTRSGL
TCSMWNKNME DLHRHIFWEP DASKLNENYC RNPDDDAHGP 451 WCYTGNPLIP
WDYCPISRCE GDTTPTIVNL DHPVISCAKT KQLRVVNGIP 501 TRTNVGWMIS
LRYRNKHICG GSLIKESWVL TARQCFPSRD LKDYEAWLGI 551 HDVHGRGEEK
RKQVLNVSQL VYGPEGSDLV LMKLARPAVL DDFVNTIDLP 601 NYGCTIPEKT
SCSVYGWGYT GLINYDGLLR VAHLYIMGNE KCSQHHRGKV 651 TLNESEICAG
AEKIGSGPCE GDYGGPLVCE QHKMRMVLGV IVPGRGCAIP 701 NRPGIFVRVA
YYAKWIHKII LTYKVPES Human HGFR (SEQ ID NO:5) 1 MKAPAVLAPG
ILVLLFTLVQ RSNGECKEAL AKSEMNVNMK YQLPNFTAET 51 PIQNVILHEH
HIFLGATNYI YVLNEEDLQK VAEYKTGPVL EHPDCFPCQD 101 CSSKANLSGG
VWKDNINMAL VVDTYYDDQL ISCGSVNRGT CQRHVFPHNH 151 TADIQSEVHC
IFSPQIEEPS QCPDCVVSAL GAKVLSSVKD RFINFFVGNT 201 INSSYFPDHP
LHSISVRRLK ETKDGFMFLT DQSYIDVLPE FRDSYPIKYV 251 HAFESNNFIY
FLTVQRETLD AQTFHTRIIR FCSINSGLHS YMEMPLECIL 301 TEKRKKRSTK
KEVFNILQAA YVSKPGAQLA RQIGASLNDD ILFGVFAQSK 351 PDSAEPMDRS
ANCAFPIKYV NDFFNKIVNK NNVRCLQHFY GPNHEHCFNR 401 TLLRNSSGCE
ARRDEYRTEF TTALQRVDLF MGQFSEVLLT SISTFIKGDL 451 TIANLGTSEG
RFMQVVVSRS GPSTPHVNFL LDSHPVSPEV IVEHTLNQNG 501 YTLVITGKKI
TKIPLNGLGC RHFQSCSQCL SAPPFVQCGW CHDKCVRSEE 551 CLSGTWTQQI
CLPAIYKVFP NSAPLEGGTR LTICGWDFGF RRNNKFDLKK 601 TRVLLGNESC
TLTLSESTMN TLKCTVGPAM NKHFNMSIII SNGHGTTQYS 651 TFSYVDPVIT
SISPKYGPMA GGTLLTLTGN YLNSGNSRHI SIGGKTCTLK 701 SVSNSILECY
TPAQTISTEF AVKLKIDLAN RETSIFSYRE DPIVYEIHPT 751 KSFISGGSTI
TGVGKNLNSV SVPRMVINVH EAGRNFTVAC QHRSNSEIIC 801 CTTPSLQQLN
LQLPLKTKAF FMLDGILSKY FDLIYVHNPV FKPFEKPVMI 851 SMGNENVLEI
KGNDIDPEAV KGEVLKVGNK SCENIHLHSE AVLCTVPNDL 901 LKLNSELNIE
WKQAISSTVL GKVIVQPDQN FTGLIAGVVS ISTALLLLLG 951 FFLWLKKRKQ
IKDLGSELVR YDARVHTPHL DRLVSARSVS PTTEMVSNES 1001 VDYRATFPED
QFPNSSQNGS CRQVQYPLTD MSPILTSGDS DISSPLLQNT 1051 VHIDLSALNP
ELVQAVQHVV IGPSSLIVHF NEVIGRGHFG CVYHGTLLDN 1101 DGKKIHCAVK
SLNRITDIGE VSQFLTEGII MKDFSHPNVL SLLGICLRSE 1151 GSPLVVLPYM
KHGDLRNFIR NETHNPTVKD LIGFGLQVAK GMKYLASKKF 1201 VHRDLAARNC
MLDEKFTVKV ADFGLARDMY DKEYYSVHNK TGAKLPVKWM 1251 ALESLQTQKF
TTKSDVWSFG VLLWELMTRG APPYFDVNTF DITVYLLQGR 1301 RLLQPEYCPD
PLYEVMLKCW HPKAEMRPSF SELVSRISAI FSTFIGEHYV 1351 HVNATYVNVK
CVAPYPSLLS SEDNADDEVD TRPASFWETS Chimpanzee HGFR (SEQ ID NO:6) 1
MKAPAVLAFG ILVLLFTLVQ RSNGECKEAL AKSEMNVNMK YQLFNFTAET 51
PIQNVILHEH HIFLGATNYI YVLNEEDLQK VAEYKTGPVL EHPDCFPCQD 101
CSSKANLSGG VWKDNINMAL VVDTYYDDQL ISCGSVNRGT CQRHVFPHNH 151
TADIQSEVHC IFSPQIEEPS QCPDCVVSAL GAKVLSSVKD RFINFFVGNT 201
INSSYFPDHP LHSISVRRLK ETKDGFMFLT DQSYIDVLPE FRDSYPIKYV 251
HAFESNNFIY FLTVQRETLD AQTFHTRIIR FCSINSGLHS YMEMPLECIL 301
TEKRKKRSTK KEVFNILQAA YVSKPGAQLA RQIGASLNDD ILFGVFAQSK 351
PDSAEPMDRS ANCAFPIKYV NDFFNKIVNK NNVRCLQHFY GPNHEHCFNR 401
TLLRNSSSCE ARRDEYRTEF TTALQRVDLF MGQFSEVLLT SISTFIKGDL 451
TIANLGTSEG RFMQVVVSRS GPSTPHVNFL LDSHPVSPEV IVEHTLNQNG 501
YTLVVTGKKI TKIPLNGLGC RHFQSCSQCL SAPPFVQCGW CHDKCVRSEE 551
CLSGTWTQQI CLPAIYKVFP NSAPLEGGTR LTICGWDFGF RRNNKFDLKK 601
TRVLLGNESC TLTLSESTMN TLKCTVGPAN NKHFNMSIII SNGHGTTQYS 651
TFSYVDPVIT SISPKYGFMA GGTLLTLTGN YLNSGNSRHI SIGGKTCTLK 701
SVSNSILECY TPAQTISTEF AVKLKIDLAN RETSIFSYRE DPIVYEIHPT 751
KSFISTWWKE PLNIVSFLFC FASGGSTITG VGKNLNSVSV PRMVINVHEA 801
GRNFTVACQH RSNSEIICCT TPSLQQLNLQ LPLKTKAFFM LDGILSKYFD 851
LIYVHNPVFK PFEKPVMISM GNENVLEIKG NDIDPEAVKG EVLKVGNKSC 901
ENIHLHSEAV LCTVPNDLLK LNSELNIEWK QAISSTVLGK VIVQPDQNFT 951
GLIAGVVSIS IALLLLLGFF LWLKKRKQIK DLGSELVRYD ARVHTPHLDR 1001
LVSARSVSPT TEMVSNESVD YRATFPEDQF PNSSQNGSCR QVQYPLTDMS 1051
PILTSGDSDI SSPLLQNTVH IDLSALNPEL VQAVQHVVIG PSSLIVHFNE 1101
VIGRGHFGCV YHGTLLDNDG KKIHCAVKSL NRITDIGEVS QFLTEGIIMK 1151
DFSHPNVLSL LGICLRSEGS PLVVLPYMKH GDLRNFIRNE THNPTVKDLI 1201
GFGLQVAKGM KYLASKKFVH RDLAARNCML DEKFTVKVAD FGLARDMYDK 1251
EYYSVHNKTG AKLPVKWMAL ESLQTQKFTT KSDVWSFGVL LWELMTRGAP 1301
PYPDVNTFDI TVYLLQGRRL LQPEYCFDPL YEVMLKCWHP KAEMRPSFSE 1351
LVSRISAIFS TFIGEHYVHV NATYVNVKCV APYPSLLSSE DNADDEVDTR 1401
PASFWETS Mouse HGFR (SEQ ID NO:7) 1 MKAPTVLAPG ILVLLLSLVQ
RSHGECKEAL VKSEMNVNMK YQLPNFTAET
51 PIQNVVLHGH HIYLGATNYI YVLNDKDLQK VSEFKTGPVL EHPDCLPCRD 101
CSSKANSSGG VWKDNINMAL LVDTYYDDQL ISCGSVNRGT CQRHVLPPDN 151
SADIQSEVHC MFSPEEESGQ CPDCVVSALG AKVLLSEKDR FINFFVGNTI 201
NSSYPPGYSL HSISVRRLKE TQDGFKFLTD QSYIDVLPEF LDSYFIKYIH 251
AFESNHFIYF LTVQKETLDA QTFHTRIIRF CSVDSGLHSY MEMPLECILT 301
EKRRKRSTRE EVFNILQAAY VSKPGANLAK QIGASPSDDI LFGVFAQSKP 351
DSAEPVNRSA VCAFPIKYVN DFFNKIVNKN NVRCLQHFYG PNHEHCFNRT 401
LLRNSSGCEA RSDEYRTEFT TALQRVDLFM GRLNQVLLTS ISTFIKGDLT 451
IANLGTSEGR FMQVVLSRTA HLTPHVNFLL DSHPVSPEVI VEHPSNQNGY 501
TLVVTGKKIT KIPLNGLGCG HFQSCSQCLS APYFIQCGWC HNQCVRFDEC 551
PSGTWTQEIC LPAVYKVFPT SAPLEGGTVL TICGWDFGFR KNNKFDLRKT 601
KVLLGNESCT LTLSESTTNT LKCTVGPANS EHFNVSVIIS NSRETTQYSA 651
FSYVDPVITS ISPRYGPQAG GTLLTLTGKY LNSGNSRHIS IGGKTCTLKS 701
VSDSILECYT PAQTTSDEFP VKLKIDLANR ETSSFSYRED PVVYEIHPTK 751
SFISGGSTIT GIGKTLNSVS LPKLVIDVHE VGVNYTVACQ HRSNSEIICC 801
TTPSLKQLGL QLPLKTKAFF LLDGILSKHF DLTYVHNPVF EPFEKPVMIS 851
MGNENVVEIK GNNIDPEAVK GEVLKVGNQS CESLHWHSGA VLCTVPSDLL 901
KLNSELNIEW KQAVSSTVLG KVIVQPDQNF AGLIIGAVSI SVVVLLLSGL 951
FLWMRKRKHK DLGSELVRYD ARVHTPHLDR LVSARSVSPT TEMVSNESVD 1001
YRATFPEDQF PNSSQNGACR QVQYPLTDLS PILTSGDSDI SSPLLQNTVH 1051
IDLSALNPEL VQAVQHVVIG PSSLIVHFNE VIGRGHFGCV YHGTLLDNDG 1101
KKIHCAVKSL NRITDIEEVS QFLTEGIIMK DFSHPNVLSL LGICLRSEGS 1151
PLVVLPYMKH GDLRNFIRNE THNPTVKDLI GFGLQVAKGM KYLASKKFVH 1201
RDLAARNCML DEKFTVKVAD FGLARDMYDK EYYSVHNKTG AKLPVKWMAL 1251
ESLQTQKFTT KSDVWSFGVL LWELMTRGAP PYPDVNTFDI TIYLLQGRRL 1301
LQPEYCPDAL YEVMLKCWHP KAEMRPSFSE LVSRISSIFS TFIGEHYVHV 1351
NATYVNVKCV APYPSLLPSQ DNIDGEGNT Rat HGFR (SEQ ID NO:8) 1 MKAPTALAPG
ILLLLLTLAQ RSHGECKEAL VKSEMNVNMK YQLPNFTAET 51 PIQNVVLHGH
HIYLGATNYI YVLNDKDLQK VSEFKTGPVV EHPDCFPCQD 101 CSSKANVSGG
VWKDNVNMAL LVDTYYDDQL ISCGSVNRGT CQRHVLPPDN 151 AADIQSEVHC
MFSPLAEEES GQCPDCVVSA LGAKVLLSEK DRFINFFVGN 201 TINSSYPPDY
SLHSISVRRL KETQDGFKFL TDQSYIDVLG EFRDSYPIKY 251 IHAFESNHFI
YFLTVQKETL DAQTFHTRII RFCSVDSGLH SYMEMPLECI 301 LTEKRRKRST
REEVFNILQA AYVSKPGANL AKQIGASPYD DILYGVFAQS 351 KPDSAEPMNR
SAVCAFPIKY VNDFFNKIVN KNNVRCLQHF YGPNHEHCFN 401 RTLLRNSSGC
EVRSDEYRTE FTTALQAVDL FMGRLNHVLL TSISTFIKGD 451 LTIANLGTSE
GRFMQVVLSR TAHFTPHVNF LLDSHPVSPE VIVEHPSNQN 501 GYTLVVTGKK
ITKIPLNGLG CGHFQSCSQC LSAPYFIQCG WCHNRCVHSN 551 ECPSGTWTQE
ICLPAVYKVF PTSAPLEGGT MLTICGWDFG FKKNNKFDLR 601 KTKVLLGNES
CTLTLSESTT NTLKCTVGPA MSEHFNVSVI VSNSRETTQY 651 SAFSYVDPVI
TSISPRYGPH AGGTLLTLTG KYLNSGNSRH ISIGGKTCTL 701 KSVSDSILEC
YTPGHTVSAE FPVKLKIDLA DRVTSSFSYG EDPFVSEIHP 751 TKSFISGGST
ITGIGKNLNS VSTPKLVIEV HDVGVNYTVA CQHRSSSEII 801 CCTTPSLQQL
DLQLPLKTKA FFLLDGILSK HFDLTYVHDP MFKPFEKPVM 851 ISMGNENVVE
IKGDDIDPEA VKGEVLKVGN KSCENLHWHS EALLCTVPSD 901 LLKLNGGELN
IEWKQAVSST VLGKVIVQPD QNFAGLIIGA VSISVVVLLV 951 SGLFLWLRKR
KHKDLGSELV RYDARVHTPH LDRLVSARSV SPTTEMVSNE 1001 SVDYRATFPE
DQFPNSSQNG ACRQVQYPLT DLSPILTSGD SDISSPLLQN 1051 TVHIDLSALN
PELVQAVQHV VIGPSSLIVH FNEVIGRGHF GCVYHGTLLD 1101 SDGKKIHCAV
KSLNRITDIE EVSQFLTEGI IMKDFSHPNV LSLLGICLRS 1151 EGSPLVVLPY
MKHGDLRNFI RNETHNPTVK DLIGFGLQVA KGMKYLASKK 1201 FVHRDLAARN
CMLDEKFTVK VADFGLARDM YDKEYYSVHN KTGAKLPVKW 1251 MALESLQTQK
FTTKSDVWSF GVLLWELMTR GAPPYPDVNT FDITIYLLQC 1301 RRLLQPEYCP
DALYEVMLKC WHPKAEMRPS FSELVSRISS IFSTFIGEHY 1351 VHVNATYVNV
KCVAPYPSLL PSQDNIDGEA NT
[0042] Exemplary regions of HGFR include: TABLE-US-00002 Region
52..487 /region_name="semaphorin domain" /note="Sema"
/db_xref="CDD:25341" Region 519..561 /region_name="domain found in
Plexins, Semaphorins and Integrins" /note="PSI"
/db_xref="CDD:25325" Region 563..656 /region_name="First repeat of
the IPT domain of Plexins and Cell Surface Receptors (PCSR)"
/note="IPT_plexin_repeat1" /db_xref="CDD:27712" Region 657..740
/region_name="Second repeat of the IPT domain of Plexins and Cell
Surface Receptors (PCSR)" /note="IPT_plexin_repeat2"
/db_xref="CDD:27711" Region 742..837 /region_name="Third repeat of
the IPT domain of Plexins and Cell Surface Receptors (PCSR)"
/note="IPT_plexin_repeat3" /db_xref="CDD:27713" Region 839..932
/region_name="IPT domain of Plexins and Cell Surface Receptors
(PCSR) and related proteins" /note="IPT_PCSR"
/db_xref="CDD:27705"
[0043] In some embodiments, HGF, and biologically active fragments
thereof are provided as purified polypeptides. Purified
polypeptides include polypeptides that are generated in vitro
(e.g., by in vitro translation or by use of an automated
polypeptide synthesizer) and polypeptides that are initially
expressed in a cell (e.g., a prokaryotic cell, a eukaryotic cell,
an insect cell, a yeast cell, a mammalian cell, a plant cell) and
subsequently purified. Cells that express a purified polypeptide
can include cells that encode an endogenous gene, cells transduced
with an expression vector encoding a polypeptide, and cells that
are experimentally manipulated to induce expression of an
endogenous gene that is not typically expressed in that cell type
(e.g., gene activation technology). In some embodiments,
polypeptides are fusion proteins (e.g., an
HGFR-glutathione-S-transferase fusion) that may include a protease
cleavage site to allow cleavage and separation of the fusion
protein into separate polypeptides. In some embodiments, a
polypeptide can include an amino acid sequence that facilitates
purification of the polypeptide (e.g., a multiple histidine tag, a
FLAG tag, etc). Methods for isolating proteins from cells or
polypeptides that are expressed by cells, include affinity
purification, size exclusion chromatography, high performance
liquid chromatography, and other chromatographic purification
methods. The polypeptides can be post-translationally modified,
e.g., glycosylated.
[0044] Purified HGF (e.g., purified human HGF) can be obtained from
a mammalian cell line stably transfected with a cDNA encoding the
human HGF polypeptide listed herein as SEQ ID NO:2 and secreting
mature HGF as described, e.g., in U.S. Pat. No. 5,686,292 to
Schwall, or Naka et al., Journal of Biol. Chem.,
267(28):20114-20119, (1992). In some embodiments, HGF can be a
single chain variant that lacks mitogenic activity but retains high
affinity receptor binding, as described in Lokker et al, EMBO J.,
11(7):2503-2510, (1992).
II. HGF and HGFR HGF/HGFR Modulators
[0045] A variety of agents can be used as a HGF/HGFR modulator to
treat pathologies related to the lymphatic system, e.g., induced
lymphedema, lymphangiomas, tumor lymphangiogenesis, or tumor
metastasis. The agent may be any type of compound that can be
administered to a subject (e.g., antibodies, proteins, peptides,
glycoproteins, glycopeptides, glycolipids, polysaccharides,
oligosaccharides, nucleic acids, bioorganic molecules,
peptidomimetics, pharmacological agents and their metabolites,
transcriptional and translation control sequences, and the like).
In one embodiment, the HGF/HGFR modulator is a biologic, e.g., a
protein having a molecular weight of between 5-300 kDa.
[0046] For example, a HGF/HGFR modulator may inhibit binding of HGF
to an HGFR or may prevent HGF-mediated NF-.kappa.B activation. A
typical HGF/HGFR modulator can bind to HGFR, e.g., a single chain
variant of HGF that lacks mitogenic activity but retains high
affinity receptor-binding (see, e.g., Lokker et al, EMBO J., 11
(7):2503-2510, (1992)). A HGF/HGFR modulator that binds to HGF may
alter the conformation of HGF, hinder binding of HGF to HGFR, or
otherwise decrease the affinity of HGF for a HGFR or prevent the
interaction between HGF and a HGFR.
[0047] A HGF/HGFR modulator (e.g., an antibody) may bind to HGF or
to a HGFR with a K.sub.d of less than 10.sup.-6, 10.sup.-7,
10.sup.-8, 10.sup.-9, or 10.sup.-10 M. In one embodiment, the
HGF/HGFR modulator binds to HGF with an affinity at least 5, 10,
20, 50, 100, 200, 500, or 1000 better than its affinity for
hepatocyte growth factor-like/macrophage stimulating protein
(HGF1/MSP). In one embodiment, the HGF/HGFR modulator binds to HGF
or HGFR with an affinity at least 5, 10, 20, 50, 100, 200, 500, or
1000-fold better than its affinity for the macrophage stimulating 1
receptor (RON) (e.g., NP.sub.--002438). A preferred HGF/HGFR
modulator specifically binds HGF or HGFR, such as a HGF or HGFR
specific antibody.
[0048] Exemplary HGF protein molecules include human HGF (e.g.,
NP.sub.--001010932, shown as SEQ ID NO:1)), Chimpanzee HGF (e.g.,
XP.sub.--519174, shown as SEQ ID NO:2), mouse HGF (e.g., CAA51054,
shown as SEQ ID NO:3), and Rat HGF (e.g., 1602237A, shown as SEQ ID
NO:4). Also included are proteins that include an amino acid
sequence at least 90, 92, 95, 97, 98, 99% identical and completely
identical to the mature processed region of the aforementioned HGF
proteins (e.g., an amino acid sequence at least 90, 92, 95, 97, 98,
99% identical or completely identical to amino acids 25-1390 of SEQ
ID NO:1 and proteins encoded by a nucleic acid that hybridizes
under high stringency conditions to a human, chimp, mouse, or rat
gene encoding a naturally occurring HGF protein. Preferably, a HGF
protein, in its processed mature form, is capable of providing at
least one HGF activity, e.g., binding to HGFR.
[0049] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). The optimal alignment is determined as
the best score using the GAP program in the GCG software package
with a Blossum 62 scoring matrix with a gap penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences.
[0050] As used herein, the term "hybridizes under high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous
and nonaqueous methods are described in that reference and either
can be used. High stringency hybridization conditions include
hybridization in 6.times.SSC at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C., or
substantially similar conditions.
[0051] Exemplary HGF/HGFR modulators include antibodies that bind
to HGF or HGFR and soluble forms of the HGFR that compete with cell
surface HGFR for binding to HGF. An example of a soluble form of
the HGFR is an Fc fusion protein that includes at least a portion
of the extracellular domain of HGFR (e.g., a soluble HGF-binding
fragment of HGFR), referred to as HGFR-Fc (see e.g., Mark et al.,
Journal of Biol. Chem., 267(36):26166-26171, (1992)). Other soluble
forms of HGFR, e.g., forms that do not include an Fc domain, can
also be used. Antibody HGF/HGFR modulators are further discussed
below. Other types of HGF/HGFR modulators, e.g., small molecules,
nucleic acid or nucleic acid-based aptamers, and peptides, can be
isolated by screening, e.g., as described in Jhaveri et al. Nat.
Biotechnol. 18:1293 and U.S. Pat. No. 5,223,409. Exemplary assays
for determining if an agent binds to HGF or HGFR and for
determining if an agent modulates a HGF/HGFR interaction are
described, e.g., in U.S. Pat. No. 6,468,529 to Schwall et al.
[0052] An exemplary soluble form of the HGFR protein includes a
region of the HGFR protein that binds to HGF, e.g., an
extracellular domain, e.g., domain of in the extracellular region.
This region can be physically associated, e.g., fused to another
amino acid sequence, e.g., an Fc domain, at its N- or C-terminus.
The region from HGFR can be spaced by a linker from the
heterologous amino acid sequence. Michieli et al. (2005), Cancer
Cell, 6:61-73, describes an exemplary HGFR fusion protein.
A. Antibodies
[0053] Exemplary HGF/HGFR modulators include antibodies that bind
to HGF and/or HGFR. In one embodiment, the antibody inhibits the
interaction between HGF and a HGFR, e.g., by physically blocking
the interaction, decreasing the affinity of HGF and/or HGFR for its
counterpart, disrupting or destabilizing HGF complexes,
sequestering HGF or a HGFR, or targeting HGF or HGFR for
degradation. In one embodiment, the antibody can bind to HGF or
HGFR at an epitope that includes one or more amino acid residues
that participate in the HGF/HGFR binding interface. Such amino acid
residues can be identified, e.g., by alanine scanning. In another
embodiment, the antibody can bind to residues that do not
participate in the HGF/HGFR binding. For example, the antibody can
alter a conformation of HGF or HGFR and thereby reduce binding
affinity, or the antibody may sterically hinder HGF/HGFR binding.
The antibody may bind to the .alpha. subunit or the .beta. subunit
of HGF.
[0054] In addition to antibodies that bind to HGF and/or HGFR,
other antibodies can be used. In one embodiment, the antibody can
prevent activation of a HGF/HGFR mediated event or activity. For
example, it is possible to use an antibody to .alpha.9 integrin,
which is upregulated by HGF. For example, certain antibodies to
.alpha.9 can inhibit HGF induced migration of cells.
[0055] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable region, e.g., an
amino acid sequence that provides an immunoglobulin variable domain
or an immunoglobulin variable domain sequence. For example, an
antibody can include a heavy (H) chain variable region (abbreviated
herein as VH), and a light (L) chain variable region (abbreviated
herein as VL). In another example, an antibody includes two heavy
(H) chain variable regions and two light (L) chain variable
regions. The term "antibody" encompasses antigen-binding fragments
of antibodies (e.g., single chain antibodies, Fab fragments,
F(ab').sub.2 fragments, Fd fragments, Fv fragments, and dAb
fragments) as well as complete antibodies, e.g., intact and/or full
length immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3,
IgG4), IgE, IgD, IgM (as well as subtypes thereof). The light
chains of the immunoglobulin may be of types kappa or lambda. In
one embodiment, the antibody is glycosylated. An antibody can be
functional for antibody-dependent cytotoxicity and/or
complement-mediated cytotoxicity, or may be non-functional for one
or both of these activities.
[0056] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
("CDR"), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the FRs and CDRs has been
precisely defined (see, e.g., Kabat, E. A., et al. (1991) Sequences
of Proteins of Immunological Interest, Fifth Edition, US Department
of Health and Human Services, NIH Publication No. 91-3242; and
Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat
definitions are used herein. Each VH and VL is typically composed
of three CDRs and four FRs, arranged from amino-terminus to
carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0057] An "immunoglobulin domain" refers to a domain from the
variable or constant domain of immunoglobulin molecules.
Immunoglobulin domains typically contain two .beta.-sheets formed
of about seven .beta.-strands, and a conserved disulfide bond (see,
e.g., A. F. Williams and A. N. Barclay (1988) Ann. Rev Immunol.
6:381-405). An "immunoglobulin variable domain sequence" refers to
an amino acid sequence that can form a structure sufficient to
position CDR sequences in a conformation suitable for antigen
binding. For example, the sequence may include all or part of the
amino acid sequence of a naturally occurring variable domain. For
example, the sequence may omit one, two, or more N- or C-terminal
amino acids, internal amino acids, may include one or more
insertions or additional terminal amino acids, or may include other
alterations. In one embodiment, a polypeptide that includes an
immunoglobulin variable domain sequence can associate with another
immunoglobulin variable domain sequence to form a target binding
structure (or "antigen binding site"), e.g., a structure that
interacts with HGF or HGFR.
[0058] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains. The heavy and light
immunoglobulin chains can be connected by disulfide bonds. The
heavy chain constant region typically includes three constant
domains, CH1, CH2, and CH3. The light chain constant region
typically includes a CL domain. The variable region of the heavy
and light chains contains a binding domain that interacts with an
antigen. The constant regions of the antibodies typically mediate
the binding of the antibody to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system.
[0059] One or more regions of an antibody can be human, effectively
human, or humanized. For example, one or more of the variable
regions can be human or effectively human. For example, one or more
of the CDRs, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and
LC CDR3, can be human. Each of the light chain CDRs can be human.
HC CDR3 can be human. One or more of the framework regions can be
human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one
embodiment, all the framework regions are human, e.g., derived from
a human somatic cell, e.g., a hematopoietic cell that produces
immunoglobulins or a non-hematopoietic cell. In one embodiment, the
human sequences are germline sequences, e.g., encoded by a germline
nucleic acid. One or more of the constant regions can be human,
effectively human, or humanized. In another embodiment, at least
70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g.,
FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4,
collectively) or the entire antibody can be human, effectively
human, or humanized. For example, FR1, FR2, and FR3 collectively
can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical,
or completely identical, to a human sequence encoded by a human
germline segment.
[0060] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. An "effectively human" antibody is an antibody that includes
a sufficient number of human amino acid positions such that the
antibody does not elicit an immunogenic response in a normal
human.
[0061] A "humanized" immunoglobulin variable region is an
immunoglobulin variable region that is modified such that the
modified form elicits less of an immune response in a human than
does the non-modified form, e.g., is modified to include a
sufficient number of human framework amino acid positions such that
the immunoglobulin variable region does not elicit an immunogenic
response in a normal human. Descriptions of "humanized"
immunoglobulins include, for example, U.S. Pat. Nos. 6,407,213 and
5,693,762. In some cases, humanized immunoglobulins can include a
non-human amino acid at one or more framework amino acid
positions.
[0062] Antibodies that bind to HGF or a HGFR can be generated by a
variety of means, including immunization, e.g., using an animal, or
in vitro methods such as phage display. All or part of HGF or HGFR
can be used as an immunogen or as a target for selection. For
example, HGF or a fragment thereof or HGFR or a fragment thereof,
can be used as an immunogen. In one embodiment, the immunized
animal contains immunoglobulin producing cells with natural, human,
or partially human immunoglobulin loci. In one embodiment, the
non-human animal includes at least a part of a human immunoglobulin
gene. For example, it is possible to engineer mouse strains
deficient in mouse antibody production with large fragments of the
human Ig loci. Accordingly, by using hybridoma technology, at least
partly human, antigen-specific monoclonal antibodies with the
desired specificity can be produced and selected. See, e.g.,
XENOMOUSE.TM., Green et al. (1994) Nat. Gen. 7:13-21; US
2003-0070185; U.S. Pat. No. 5,789,650; and WO 96/34096.
[0063] Non-human antibodies to HGF and HGFR can also be produced,
e.g., in a rodent. The non-human antibody can be humanized, e.g.,
as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761;
and 6,407,213, deimmunized, or otherwise modified to make it
effectively human.
[0064] EP 239 400 (Winter et al.) describes altering antibodies by
substitution (within a given variable region) of their
complementarity determining regions (CDRs) for one species with
those from another. Typically, CDRs of a non-human (e.g., murine)
antibody are substituted into the corresponding regions in a human
antibody by using recombinant nucleic acid technology to produce
sequences encoding the desired substituted antibody. Human constant
region gene segments of the desired isotype (usually gamma I for CH
and kappa for CL) can be added and the humanized heavy and light
chain genes can be co-expressed in mammalian cells to produce
soluble humanized antibody. Other methods for humanizing antibodies
can also be used. For example, other methods can account for the
three dimensional structure of the antibody, framework positions
that are in three dimensional proximity to binding determinants,
and immunogenic peptide sequences. See, e.g., WO 90/07861; U.S.
Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101; Tempest
et al. (1991) Biotechnology 9:266-271 and U.S. Pat. No.
6,407,213.
[0065] Fully human monoclonal antibodies that bind to HGF and HGFR
can be produced, e.g., using in vitro-primed human splenocytes, as
described by Boerner et al. (1991) J. Immunol. 147:86-95. They may
be prepared by repertoire cloning as described by Persson et al.
(1991) Proc. Nat. Acad. Sci. USA 88:2432-2436 or by Huang and
Stollar (1991) J. Immunol. Methods 141:227-236; also U.S. Pat. No.
5,798,230. Large nonimmunized human phage display libraries may
also be used to isolate high affinity antibodies that can be
developed as human therapeutics using standard phage technology
(see, e.g., Hoogenboom et al. (1998) Immunotechnology 4:1-20;
Hoogenboom et al. (2000) Immunol Today 2:371-378; and US
2003-0232333).
[0066] Antibodies and other proteins described herein can be
produced in prokaryotic and eukaryotic cells. In one embodiment,
the antibodies (e.g., scFv's) are expressed in a yeast cell such as
Pichia (see, e.g., Powers et al. (2001) J. Immunol. Methods
251:123-35), Hanseula, or Saccharomyces.
[0067] Antibodies, particularly full length antibodies, e.g., IgGs,
can be produced in mammalian cells. Exemplary mammalian host cells
for recombinant expression include Chinese Hamster Ovary (CHO
cells) (including dihydrofolate reductase-negative CHO cells,
described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA
77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),
lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS
cells, K562, and a cell from a transgenic animal, e.g., a
transgenic mammal. For example, the cell can be a mammary
epithelial cell.
[0068] In addition to the nucleic acid sequence encoding the
immunoglobulin domain, the recombinant expression vectors may carry
additional nucleic acid sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and
5,179,017). Exemplary selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0069] In an exemplary system for recombinant expression of an
antibody (e.g., a full length antibody or an antigen-binding
portion thereof), a recombinant expression vector encoding both the
antibody heavy chain and the antibody light chain is introduced
into dhfr-CHO cells by calcium phosphate-mediated transfection.
Within the recombinant expression vector, the antibody heavy and
light chain genes are each operatively linked to enhancer/promoter
regulatory elements (e.g., derived from SV40, CMV, adenovirus and
the like, such as a CMV enhancer/AdMLP promoter regulatory element
or an SV40 enhancer/AdMLP promoter regulatory element) to drive
high levels of transcription of the genes. The recombinant
expression vector also carries a DHFR gene, which allows for
selection of CHO cells that have been transfected with the vector
using methotrexate selection/amplification. The selected
transformant host cells are cultured to allow for expression of the
antibody heavy and light chains and intact antibody is recovered
from the culture medium. Standard molecular biology techniques are
used to prepare the recombinant expression vector, to transfect the
host cells, to select for transformants, to culture the host cells,
and to recover the antibody from the culture medium. For example,
some antibodies can be isolated by affinity chromatography with a
Protein A or Protein G.
[0070] Antibodies (and Fc fusions) may also include modifications,
e.g., modifications that alter Fc function, e.g., to decrease or
remove interaction with an Fc receptor or with C1q, or both. For
example, the human IgG1 constant region can be mutated at one or
more residues, e.g., one or more of residues 234 and 237, e.g.,
according to the numbering in U.S. Pat. No. 5,648,260. Other
exemplary modifications include those described in U.S. Pat. No.
5,648,260.
[0071] For some proteins that include an Fc domain, the
antibody/protein production system may be designed to synthesize
antibodies or other proteins in which the Fc region is
glycosylated. For example, the Fc domain of IgG molecules is
glycosylated at asparagine 297 in the CH2 domain. The Fc domain can
also include other eukaryotic post-translational modifications. In
other cases, the protein is produced in a form that is not
glycosylated.
[0072] Antibodies and other proteins can also be produced by a
transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a
method for expressing an antibody in the mammary gland of a
transgenic mammal. A transgene is constructed that includes a
milk-specific promoter and nucleic acid sequences encoding the
antibody of interest, e.g., an antibody described herein, and a
signal sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the protein of
interest, e.g., an antibody or Fc fusion protein. The protein can
be purified from the milk, or for some applications, used
directly.
[0073] Methods described in the context of antibodies can be
adapted to other proteins, e.g., Fc fusions and soluble receptor
fragments.
B. Variants of HGF
[0074] In some embodiments, HGF proteins can be HGF variants that
are resistant to proteolytic cleavage by enzymes that are capable
of in vivo conversion of HGF into its two-chain form. The variants
are preferably stabilized in single-chain form by site directed
mutagenesis within a region recognized by an enzyme capable of
converting HGF into its two-chain form. These HGF variants retain
substantially full receptor binding affinity of the corresponding
wild-type HGF, but do not activate the HGFR. In one embodiment, HGF
variants can have enhanced receptor binding affinity relative to
the corresponding wild-type HGF but be unable to activate the HGFR.
Such compounds are competitive antagonists of the corresponding
wild-type HGF and, when present in sufficient concentration, are
capable of inhibiting the binding of wild-type HGF to HGFR. See,
e.g., Lokker et al, EMBO J., 11(7):2503-2510, (1992) and U.S. Pat.
No. 5,316,921 to Godowski et al. Accordingly they can be used as
HGF/HGFR antagonists.
C. Peptides
[0075] In some embodiments, the HGF/HGFR modulator can be a peptide
of 32 amino acids or less that independently binds to but does not
activate a target molecule (e.g., HGFR). Some such peptides can
include one or more disulfide bonds. Other peptides, so-called
"linear peptides," are devoid of cysteines. In one embodiment, the
peptides are artificial, i.e., not present in Nature or not present
in a protein encoded by one or more genomes of interest, e.g., the
human genome. Synthetic peptides may have little or no structure in
solution (e.g., unstructured), heterogeneous structures (e.g.,
alternative conformations or "loosely structured), or a singular
native structure (e.g., cooperatively folded). Some synthetic
peptides adopt a particular structure when bound to a target
molecule. Some exemplary synthetic peptides are so-called "cyclic
peptides" that have at least a disulfide bond and, for example, a
loop of about 4 to 12 non-cysteine residues. Exemplary peptides are
less than 28, 24, 20, or 18 amino acids in length.
[0076] Peptide sequences that independently bind HGFR can be
identified by any of a variety of methods. For example, they can be
selected from a display library or an array of peptides. After
identification, such peptides can be produced synthetically or by
recombinant means. The sequences can be incorporated (e.g.,
inserted, appended, or attached) into longer sequences.
[0077] The techniques discussed in Kay et al., Phage Display of
Peptides and Proteins. A Laboratory Manual (Academic Press, Inc.,
San Diego 1996) and U.S. Pat. No. 5,223,409 are useful for
preparing a library of potential binders corresponding to the
selected parental template. Peptide display libraries can be
prepared according to such techniques, and screened for peptides
that bind to and inhibit HGFR.
[0078] In addition, phage libraries or selected populations from
phage libraries can be counter-selected, e.g., by counter-selection
with an HGFR binding domain that lacks a SEMA (semaphorin) domain
or a PSI (plexin/semaphorin/integrin) domain, both of which
contribute to HGF binding. Such procedures can be used to discard
peptides that do not contact the HGF binding site.
[0079] Peptides can also be synthesized using alternative
backbones, e.g., a peptoid backbone, e.g., to produce a compound
which has increased protease resistance. In particular this method
can be used to make a compound that binds to and inhibits
activation of HGFR and is not cleaved, e.g., by serum
proteases.
[0080] A polypeptide that inhibits HGFR activation can be
associated with (e.g., conjugated to) a polymer, e.g., a
substantially non-antigenic polymers, such as polyalkylene oxides
or polyethylene oxides. Suitable polymers will vary substantially
by weight. Polymers having molecular number average weights ranging
from about 200 to about 35,000 (or about 1,000 to about 15,000, and
2,000 to about 12,500) can be used. A plurality of polymer moieties
can be attached to one polypeptide, e.g., at least two, three, or
four such moieties, e.g., having an average molecular weight of
about 2,000 to 7,000 Daltons.
[0081] For example, the polypeptide can be conjugated to a water
soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.
polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0082] Other compounds can also be attached to the same polymer,
e.g., a cytotoxin, a label, or another targeting agent or an
unrelated agent. Mono-activated, alkoxy-terminated polyalkylene
oxides (PAO's), e.g., monomethoxy-terminated polyethylene glycols
(mPEG's); C.sub.1-4 alkyl-terminated polymers; and bis-activated
polyethylene oxides (glycols) can be used for crosslinking. See,
e.g., U.S. Pat. No. 5,951,974.
D. Nucleic Acid Antagonists
[0083] In certain implementations, nucleic acid antagonists are
used to decrease expression of an endogenous gene encoding HGF, a
HGFR, integrin (.alpha.9, or stanniocalcin 1. In one embodiment,
the nucleic acid antagonist is an siRNA that targets mRNA encoding
HGF or a HGFR. Other types of antagonistic nucleic acids can also
be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or an
antisense nucleic acid. In some embodiments, nucleic acid
antagonists can be directed to downstream effector targets of HGFR
activation (e.g., human .alpha.9 integrin, an exemplary sequence of
which is listed under Genbank No. NM.sub.--002207).
[0084] siRNAs are small double stranded RNAs (dsRNAs) that
optionally include overhangs. For example, the duplex region of an
siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20,
21, 22, 23, or 24 nucleotides in length. Typically, the siRNA
sequences are exactly complementary to the target mRNA. dsRNAs and
siRNAs in particular can be used to silence gene expression in
mammalian cells (e.g., human cells). siRNAs also include short
hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3'
overhangs. See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci.
USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA
98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et
al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al.
(2005), Nat. Biotechnol. 23(2):227-31; 20040086884; U.S.
20030166282; 20030143204; 20040038278; and 20030224432.
[0085] Anti-sense agents can include, for example, from about 8 to
about 80 nucleobases (i.e., from about 8 to about 80 nucleotides),
e.g., about 8 to about 50 nucleobases, or about 12 to about 30
nucleobases. Anti-sense compounds include ribozymes, external guide
sequence (EGS) oligonucleotides (oligozymes), and other short
catalytic RNAs or catalytic oligonucleotides which hybridize to the
target nucleic acid and modulate its expression. Anti-sense
compounds can include a stretch of at least eight consecutive
nucleobases that are complementary to a sequence in the target
gene. An oligonucleotide need not be 100% complementary to its
target nucleic acid sequence to be specifically hybridizable. An
oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target interferes with the normal function
of the target molecule to cause a loss of utility, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment
or, in the case of in vitro assays, under conditions in which the
assays are conducted.
[0086] Hybridization of antisense oligonucleotides with mRNA (e.g.,
an mRNA encoding HGF or HGFR) can interfere with one or more of the
normal functions of mRNA. The functions of mRNA to be interfered
with include all key functions such as, for example, translocation
of the RNA to the site of protein translation, translation of
protein from the RNA, splicing of the RNA to yield one or more mRNA
species, and catalytic activity which may be engaged in by the RNA.
Binding of specific protein(s) to the RNA may also be interfered
with by antisense oligonucleotide hybridization to the RNA.
[0087] Exemplary antisense compounds include DNA or RNA sequences
that specifically hybridize to the target nucleic acid, e.g., the
mRNA encoding HGF or HGFR. The complementary region can extend for
between about 8 to about 80 nucleobases. The compounds can include
one or more modified nucleobases. Modified nucleobases may include,
e.g., 5-substituted pyrimidines such as 5-iodouracil,
5-iodocytosine, and C5-propynyl pyrimidines such as
C5-propynylcytosine and C5-propynyluracil. Other suitable modified
nucleobases include N.sup.4--(C.sub.1-C.sub.12) alkylaminocytosines
and N.sup.4,N.sup.4--(C.sub.1-C.sub.12) dialkylaminocytosines.
Modified nucleobases may also include
7-substituted-8-aza-7-deazapurines and 7-substituted-7-deazapurines
such as, for example, 7-iodo-7-deazapurines,
7-cyano-7-deazapurines, 7-aminocarbonyl-7-deazapurines. Examples of
these include 6-amino-7-iodo-7-deazapurines,
6-amino-7-cyano-7-deazapurines,
6-amino-7-aminocarbonyl-7-deazapurines,
2-amino-6-hydroxy-7-iodo-7-deazapurines,
2-amino-6-hydroxy-7-cyano-7-deazapurines, and
2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore,
N.sup.6--(C.sub.1-C.sub.12) alkylaminopurines and
N.sup.6,N.sup.6--(C.sub.1-C.sub.12) dialkylaminopurines, including
N.sup.6-methylaminoadenine and
N.sup.6,N.sup.6-dimethylaminoadenine, are also suitable modified
nucleobases. Similarly, other 6-substituted purines including, for
example, 6-thioguanine may constitute appropriate modified
nucleobases. Other suitable nucleobases include 2-thiouracil,
8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and
2-fluoroguanine. Derivatives of any of the aforementioned modified
nucleobases are also appropriate. Substituents of any of the
preceding compounds may include C.sub.1-C.sub.30 alkyl,
C.sub.2-C.sub.30 alkenyl, C.sub.2-C.sub.30 alkynyl, aryl, aralkyl,
heteroaryl, halo, amino, amido, nitro, thio, sulfonyl, carboxyl,
alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like.
[0088] Descriptions of other types of nucleic acid agents are also
available. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and
5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense
RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988)
Nature 334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84;
Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992)
Bioassays 14:807-15.
E. Artificial Transcription Factors
[0089] Artificial transcription factors can also be used to
regulate expression of HGF, a HGFR, integrin .alpha.9, or
stanniocalcin 1. The artificial transcription factor can be
designed or selected from a library, e.g., for ability to bind to a
sequence in an endogenous gene encoding HGF or HGFR, e.g., in a
regulatory region, e.g., the promoter. For example, the artificial
transcription factor can be prepared by selection in vitro (e.g.,
using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by
design based on a recognition code (see, e.g., WO 00/42219 and U.S.
Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol
267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al.
(2001) Nat. Biotechnol 19:656; and Wu et al. (1995) Proc. Natl.
Acad. Sci. USA 92:344 for, among other things, methods for creating
libraries of varied zinc finger domains.
[0090] Optionally, an artificial transcription factor can be fused
to a transcriptional regulatory domain, e.g., an activation domain
to activate transcription or a repression domain to repress
transcription. In particular, repression domains can be used to
decrease expression of endogenous genes encoding HGF or HGFR. The
artificial transcription factor can itself be encoded by a
heterologous nucleic acid that is delivered to a cell or the
protein itself can be delivered to a cell (see, e.g., U.S. Pat. No.
6,534,261). The heterologous nucleic acid that includes a sequence
encoding the artificial transcription factor can be operably linked
to an inducible promoter, e.g., to enable fine control of the level
of the artificial transcription factor in the cell, e.g., an
endothelial cell.
F. Pharmaceutical Compositions
[0091] A HGF/HGFR modulator (e.g., an antibody or soluble HGFR
protein, e.g., a HGFR extracellular region fused to a Fc) can be
formulated as a pharmaceutical composition, e.g., for
administration to a subject to a pathology related to the lymphatic
system (e.g., a disorder described herein, such as lymphedema,
lymphatic filariasis, lymphangiomas, tumor lymphangiogenesis, or
tumor metastasis). Typically, a pharmaceutical composition includes
a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. The composition can include a
pharmaceutically acceptable salt, e.g., an acid addition salt or a
base addition salt (see, e.g., Berge, S. M., et al. (1977) J.
Pharm. Sci. 66:1-19).
[0092] The HGF/HGFR modulator can be formulated according to
standard methods. Pharmaceutical formulation is a well-established
art, and is further described, e.g., in Gennaro (ed.), Remington:
The Science and Practice of Pharmacy, 20.sup.th ed., Lippincott,
Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7.sup.th
Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN:
0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients
American Pharmaceutical Association, 3.sup.rd ed. (2000) (ISBN:
091733096X).
[0093] In one embodiment, the HGF/HGFR modulator (e.g., an antibody
or HGFR-Fc) can be formulated with excipient materials, such as
sodium chloride, sodium dibasic phosphate heptahydrate, sodium
monobasic phosphate, and a stabilizer. It can be provided, for
example, in a buffered solution at a suitable concentration and can
be stored at 2-8.degree. C.
[0094] The pharmaceutical compositions may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form can depend
on the intended mode of administration and therapeutic application.
Typically compositions for the agents described herein are in the
form of injectable or infusible solutions.
[0095] Such compositions can be administered by a parenteral mode
(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular
injection). The phrases "parenteral administration" and
"administered parenterally" as used herein mean modes of
administration other than enteral and topical administration,
usually by injection, and include, without limitation, intravenous,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion.
[0096] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable for stable storage at high concentration. Sterile
injectable solutions can be prepared by incorporating an agent
described herein in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating an agent described herein
into a sterile vehicle that contains a basic dispersion medium and
the required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying that yields a powder of an agent described herein
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0097] In certain embodiments, the HGF/HGFR modulator may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0098] A HGF/HGFR modulator (e.g., an antibody or soluble HGFR
protein) can be modified, e.g., with a moiety that improves its
stabilization and/or retention in circulation, e.g., in blood,
serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50
fold.
[0099] For example, the HGF/HGFR modulator (e.g., an antibody or
soluble HGFR protein) can be associated with a polymer, e.g., a
substantially non-antigenic polymer, such as a polyalkylene oxide
or a polyethylene oxide. Suitable polymers will vary substantially
by weight. Polymers having molecular number average weights ranging
from about 200 to about 35,000 Daltons (or about 1,000 to about
15,000, and 2,000 to about 12,500) can be used.
[0100] For example, a HGF/HGFR modulator can be conjugated to a
water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.,
polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; and branched or unbranched
polysaccharides.
[0101] When the HGF/HGFR modulator (e.g., an antibody or soluble
HGFR protein) is used in combination with a second agent, the two
agents can be formulated separately or together. For example, the
respective pharmaceutical compositions can be mixed, e.g., just
prior to administration, and administered together or can be
administered separately, e.g., at the same or different times.
[0102] The lymphatic vascular system plays a pivotal role in tissue
fluid homeostasis, which affects and is affected by a number of
pathologies including, e.g., cancer cell metastasis; acquired
lymphedema, e.g., induced by surgery, radiation therapy, or
infection; skin conditions, e.g., epidermolysis caused by aging or
excessive exposure to ultra-violet light. In addition, tumor
metastasis occurs primarily through the lymphatic system, and the
extent of lymph node involvement is a key prognostic factor for
severity of disease. Lymphangiogenesis and the quantity of
intratumoral lymphatic vessels in primary tumors have been
correlated with tumor metastasis in animal experiments, for
example, in breast cancer. (Skobe et al., Nature Medicine
7(2):192-198 (2001)). Intratumoral lymphatic vasculature can play
an important role in the metastasis of many tumor types such as
breast, colon, lung, thyroid, gastric, squamous cell cancers,
mesotheliomas, osteosarcomas, and neuroblastomas.
G. Administration
[0103] The HGF/HGFR modulator (e.g., an antibody or soluble HGFR
protein) or other agent can be administered to a subject, e.g., a
human subject, by a variety of methods. For many applications, the
route of administration is one of: intravenous injection or
infusion (IV), subcutaneous injection (SC), intraperitoneally (IP),
or intramuscular injection. The HGF/HGFR modulator can be
administered as a fixed dose, or a dose adjusted for the subject's
weight (e.g., a mg/kg dose).
[0104] The dose can also be chosen to reduce or avoid production of
antibodies against the HGF/HGFR modulator.
[0105] The route and/or mode of administration of the can also be
tailored for the individual case, e.g., by monitoring the subject,
e.g., using tomographic imaging, lymphangiography, and standard
parameters associated with the particular disease, e.g., criteria
for assessing lymphatic and lymphatic system-related disorders.
[0106] Dosage regimens are adjusted to provide the desired
response, e.g., a therapeutic response or a combinatorial
therapeutic effect. Generally, any combination of doses (either
separate or co-formulated) of the HGF/HGFR modulator (e.g., an
antibody) (and optionally a second agent) can be used in order to
provide a subject with the agent in bioavailable quantities. For
example, doses in the range of 1 mg/kg-100 mg/kg, 0.5-20 mg/kg, or
1-10 mg/kg can be administered.
[0107] Dosage unit form or "fixed dose" as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier and
optionally in association with the other agent.
[0108] The HGF/HGFR modulator may be administered at least once
between about 10 minutes to about 48 hours, more preferably between
about 10 minutes and 24 hours, more preferably within 3 hours,
after the onset of symptoms or manifestation of a lymphatic or
lymphatic system-related disorder. For example, the agent may be
administered to a patient suffering or at risk for lymphedema.
Single or multiple dosages may be given. Alternatively, or in
addition, the HGF/HGFR modulator agent may be administered via
continuous infusion. The treatment can continue for days, weeks,
months or even years so as to adequately modulate lymphangiogenesis
in lymphatic or lymphatic system related disorders.
[0109] The HGF/HGFR modulator can be administered one time per week
for between about 1 to 10 weeks, preferably between 2 to 8 weeks,
more preferably between about 3 to 7 weeks, and even more
preferably for about 4, 5, or 6 weeks. The skilled artisan will
appreciate that certain factors may influence the dosage and timing
required to effectively treat a subject, including but not limited
to the severity of the disease or disorder, previous treatments,
the general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a compound can include a single treatment or,
preferably, can include a series of treatments.
[0110] If a subject is at risk for developing a disorder described
herein (e.g., filariasis) or another lymphatic system-related
disorder, the HGF/HGFR modulator can be administered before the
onset of the condition as a preventative measure. The duration of
such preventative treatment can be a single dosage of the HGF/HGFR
modulator or the treatment may continue (e.g., multiple dosages),
for example, a subject at risk for a disorder described herein may
be treated with the HGF/HGFR modulator for days, weeks, months, or
even years so as to prevent the injury from occurring.
[0111] A pharmaceutical composition may include a "therapeutically
effective amount" of an agent described herein. Such effective
amounts can be determined based on the effect of the administered
agent, or the combinatorial effect of agents if more than one agent
is used. A therapeutically effective amount of an agent may also
vary according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to elicit
a desired response in the individual, e.g., amelioration of at
least one disorder parameter or amelioration of at least one
symptom of the disorder. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the composition is
outweighed by the therapeutically beneficial effects.
[0112] An antagonist of HGF/HGFR can be used to treat cancer.
Examples of cancerous disorders include, but are not limited to,
solid tumors, soft tissue tumors, and metastatic lesions. Examples
of solid tumors include malignancies, e.g., sarcomas,
adenocarcinomas, and carcinomas, of the various organ systems, such
as those affecting lung, breast, lymphoid, gastrointestinal (e.g.,
colon), and genitourinary tract (e.g., renal, urothelial cells),
pharynx, prostate, ovary as well as adenocarcinomas which include
malignancies such as most colon cancers, rectal cancer, renal-cell
carcinoma, liver cancer, non-small cell carcinoma of the lung,
cancer of the small intestine and so forth. Metastatic lesions of
the aforementioned cancers, and particularly metastatic forms of
these cancers, can also be treated or prevented using the methods
and compositions described herein.
[0113] The method can be used to treat malignancies of the various
organ systems, such as those affecting lung, breast, lymphoid,
gastrointestinal (e.g., colon), and genitourinary tract, prostate,
ovary, pharynx, as well as adenocarcinomas which include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer and/or testicular tumors, non-small cell carcinoma
of the lung, cancer of the small intestine and cancer of the
esophagus. Exemplary solid tumors that can be treated include:
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, non-small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0114] The term "carcinoma" is recognized by those skilled in the
art and refers to malignancies of epithelial or endocrine tissues
including respiratory system carcinomas, gastrointestinal system
carcinomas, genitourinary system carcinomas, testicular carcinomas,
breast carcinomas, prostatic carcinomas, endocrine system
carcinomas, and melanomas. Exemplary carcinomas include those
forming from tissue of the cervix, lung, prostate, breast, head and
neck, colon and ovary. The term also includes carcinosarcomas,
e.g., which include malignant tumors composed of carcinomatous and
sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma
derived from glandular tissue or in which the tumor cells form
recognizable glandular structures. The term "sarcoma" is recognized
by those skilled in the art and refers to malignant tumors of
mesenchymal derivation.
H. Devices and Kits
[0115] Pharmaceutical compositions that include the HGF/HGFR
modulator (e.g., an antibody or soluble HGFR) can be administered
with a medical device. The device can designed with features such
as portability, room temperature storage, and ease of use so that
it can be used in emergency situations, e.g., by an untrained
subject or by emergency personnel in the field, removed to medical
facilities and other medical equipment. The device can include,
e.g., one or more housings for storing pharmaceutical preparations
that include HGF/HGFR modulator, and can be configured to deliver
one or more unit doses of the HGF/HGFR modulator.
[0116] For example, the pharmaceutical composition can be
administered with a needleless hypodermic injection device, such as
the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851;
5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples
of well-known implants and modules include: U.S. Pat. No.
4,487,603, which discloses an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicaments through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system.
Many other devices, implants, delivery systems, and modules are
also known.
[0117] A HGF/HGFR modulator (e.g., an antibody or soluble HGFR
protein) can be provided in a kit. In one embodiment, the kit
includes (a) a container that contains a composition that includes
a HGF/HGFR modulator, and optionally (b) informational material.
The informational material can be descriptive, instructional,
marketing or other material that relates to the methods described
herein and/or the use of the agents for therapeutic benefit. In one
embodiment, the kit also includes a second agent for treating
lymphatic and lymphatic system-related disorders. For example, the
kit includes a first container that contains a composition that
includes the HGF/HGFR modulator, and a second container that
includes the second agent.
[0118] The informational material of the kits is not limited in its
form. In one embodiment, the informational material can include
information about production of the compound, molecular weight of
the compound, concentration, date of expiration, batch or
production site information, and so forth. In one embodiment, the
informational material relates to methods of administering the
HGF/HGFR modulator (e.g., an antibody or soluble HGFR protein),
e.g., in a suitable dose, dosage form, or mode of administration
(e.g., a dose, dosage form, or mode of administration described
herein), to treat a subject who has had or who is at risk for a
lymphatic or lymphatic system-related disorder. The information can
be provided in a variety of formats, include printed text, computer
readable material, video recording, or audio recording, or as
information that provides a link or address to substantive
material.
[0119] In addition to the HGF/HGFR modulator, the composition in
the kit can include other ingredients, such as a solvent or buffer,
a stabilizer, or a preservative. The HGF/HGFR modulator can be
provided in any form, e.g., liquid, dried or lyophilized form,
preferably substantially pure and/or sterile. When the agents are
provided in a liquid solution, the liquid solution preferably is an
aqueous solution. When the agents are provided in a dried form,
reconstitution generally is achieved by the addition of a suitable
solvent. The solvent, e.g., sterile water or buffer, can optionally
be provided in the kit.
[0120] The kit can include one or more containers for the
composition or compositions containing the agents. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle, vial, or
syringe, and the informational material can be contained in a
plastic sleeve or packet. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of the agents. The containers can include a
combination unit dosage, e.g., a unit that includes both the
HGF/HGFR modulator and the second agent, e.g., in a desired ratio.
For example, the kit includes a plurality of syringes, ampules,
foil packets, blister packs, or medical devices, e.g., each
containing a single combination unit dose. The containers of the
kits can be air tight, waterproof (e.g., impermeable to changes in
moisture or evaporation), and/or light-tight.
[0121] The kit optionally includes a device suitable for
administration of the composition, e.g., a syringe or other
suitable delivery device. The device can be provided pre-loaded
with one or both of the agents or can be empty, but suitable for
loading.
III. Methods of Screening for Modulators of HGF/HGFR Pathway
Activity
[0122] Screening of putative modulators of expression of HGF/HGFR
pathway (e.g., HGF, HGFR, integrin .alpha.9 and stanniocalcin 1)
can be carried out by determining the effect of the modulators on
HGF or HGFR promoter activity in vitro or in vivo. For example, a
nucleic acid that includes a HGFR promoter (e.g., of the human,
monkey or mouse HGFR gene) or regulatory region thereof, e.g.,
Gambarotta et al. (1994), J. Biol. Chem., 269(17):12852-12857 or
e.g., a HGF promoter (e.g., of the human, monkey or mouse HGF gene)
or regulatory region thereof, e.g., Bell et al. (1998), J. Biol.
Chem., 273(12):6900-6908 can be operably linked to a nucleic acid
that encodes a reporter polypeptide, e.g., one of the reporter
polypeptides described below (e.g., enhanced green fluorescent
protein). Other promoters that can be used include those of other
genes modulated by HGF/HGFR pathway activity, e.g., integrin
.alpha.9 and stanniocalcin 1. The nucleic acid including the target
promoter operably linked to the reporter nucleic acid can be
introduced into cells in culture and/or be used to generate a
transgenic animal, allowing evaluation of promoter activity in
vivo. In some embodiments, a transgenic animal can also be
evaluated for other phenotypes (e.g., induction or repression of
gene expression in skin) affected by administration of an HGF/HGFR
modulator.
A. Evaluating Effects of Putative HGF/HGFR Modulators on Skin
[0123] Methods disclosed herein allow evaluating a compound for its
effect on the expression of HGF or HGFR or other target gene in an
experimental subject. In some embodiments, the effect of a compound
on skin (e.g., a therapeutic compound for a skin condition) can be
evaluated in the same experimental subject in which expression is
determined. The effect on skin is usually determined as an effect
on the expression of a gene under the control of a
skin-metabolism-related promoter. Such promoters include those
which control the expression and/or synthesis of: a product which
is a component of the skin, e.g., the dermis or epidermis; a
product which affects hydration or nutrition of the skin; a product
which promotes the synthesis, or degradation, of components of the
skin; a product which affects the vasculature of the skin; a
product which affects hair follicle metabolism; a product which
affects skin glandular structures; a product which affects
subcutaneous musculature; a product which affects adipose tissue;
or a product which affects cutaneous nerves.
[0124] Methods of the invention are useful for evaluating a
compound for an effect on a parameter related to the appearance or
health of the skin, for example, the elasticity of the skin, the
propensity of the skin to wrinkle, the ability of the skin to
retain fluids, e.g., water or an oil, the ability of the skin to
resist or repair damage, e.g., light or UV induced damage, the
metabolism of hair follicles including growth cycling or pigment
deposition, or subcutaneous muscle tone and function. Generally,
effects on these parameters will be evaluated indirectly, e.g., by
the effect on the expression of a reporter gene under the control
of a promoter which is normally coupled to a gene which encodes a
product which affects any of the these parameters.
Transgenic Animals
[0125] Transgenic animals which can be used in the methods of the
invention include non-human mammals, such as pigs, e.g., mini-pigs;
or rodents, e.g., mice, rats, or guinea pigs, e.g., hairless mice
(described in, for example, Begona M. et al. (1994) Proc. Natl.
Acad. Sci. 91:7717-7721), nude mice, senescence accelerated mice
(described in, for example, Takeda et al. (1991) L. Am. Geriatr.
Soc. 39:911-19), or transgenic mutant mice which exhibit a
phenotype of accelerated aging. One or more, and preferably
essentially all, of the cells of the animal include a transgene.
The transgenic animals can be homozygous or heterozygous for the
transgene. Mice are a preferred subject animal.
[0126] Many methods of making transgenic animals, e.g., mice, are
known in the art. One exemplary approach is described below.
[0127] Procedures for embryo manipulation and microinjection are
described in, for example, Manipulating the Mouse Embryo (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986, the
contents of which are incorporated herein by reference). Mouse
zygotes can be collected from six week old females that have been
super ovulated with pregnant mares serum (PMS) followed 48 hours
later with human chorionic gonadotropin. Primed females are placed
with males and checked for vaginal plugs on the following morning.
Pseudo pregnant females are selected for estrus, placed with proved
sterile vasectomized males and used as recipients. Zygotes are
collected and cumulus cells removed. Furthermore, blastocysts can
be harvested. Pronuclear embryos are recovered from female mice
mated to males. Females are treated with pregnant mare serum, PMS,
to induce follicular growth and human chorionic gonadotropin, hCG,
to induce ovulation. Embryos are recovered in a Dulbecco's modified
phosphate buffered saline (DPBS) and maintained in Dulbecco's
modified essential medium (DMEM) supplemented with 10% fetal bovine
serum.
[0128] Microinjection of a transgenic construct can be performed
using standard micro manipulators attached to a microscope. For
instance, embryos are typically held in 100 microliter drops of
DPBS under oil while being microinjected. DNA solution is
microinjected into the male pronucleus. Successful injection is
monitored by swelling of the pronucleus. Recombinant ES cells can
be injected into blastocysts, using similar techniques. Immediately
after injection embryos are transferred to recipient females, e.g.
mature mice mated to vasectomized male mice. In a general protocol,
recipient females are anesthetized, paralumbar incisions are made
to expose the oviducts, and the embryos are transformed into the
ampullary region of the oviducts. The body wall is sutured and the
skin closed with wound clips.
Screening for the Presence of the Targeting Construct
[0129] Transgenic animals can be identified after birth by standard
protocols. DNA from tail tissue can be screened for the presence of
the targeting construct using southern blots and/or PCR. Offspring
that appear to be mosaics are then crossed to each other if they
are believed to carry the targeting construct in their germ line to
generate homozygous transgenic animals. If it is unclear whether
the offspring will have germ line transmission, they can be crossed
with a parental or other strain and the offspring screened for
heterozygosity. The heterozygotes are identified by southern blots
and/or PCR amplification of the DNA.
[0130] The heterozygotes can then be crossed with each other to
generate homozygous transgenic offspring. Homozygotes may be
identified by southern blotting of equivalent amounts of genomic
DNA from mice that are the product of this cross, as well as mice
that are known heterozygotes and wild type mice. Probes to screen
the southern blots can be designed as set forth above.
[0131] Other means of identifying and characterizing the transgenic
offspring are known in the art. For example, northern blots can be
used to probe the mRNA for the presence or absence of transcripts
encoding the reporter gene. In addition, western blots can be used
to assess the level of expression of the transgene in various
tissues of these offspring by probing the western blot with an
antibody against the protein encoded by the transgene, or an
antibody against the marker gene product, where this gene is
expressed. Finally, in situ analysis (such as fixing the cells and
labeling with antibody) and/or FACS (fluorescence activated cell
sorting) analysis of various cells from the offspring can be
performed using suitable antibodies to look for the presence or
absence of the transgene product. Transgenic animals can be
generated that have two separate transgenes with distinct promoters
operably linked to detectably distinct reporters, e.g., by
interbreeding mice transgenic for the individual promoter-reporter
transgenes.
[0132] Other transgenic animals can be used in methods of the
invention. Methods for the preparation of a variety of animals are
known in the art. A protocol for the production of a transgenic pig
can be found in White and Yannoutsos, Current Topics in Complement
Research: 64th Forum in Immunology, pp. 88-94; U.S. Pat. No.
5,523,226; U.S. Pat. No. 5,573,933; PCT Application WO93/25071; and
PCT Application WO95/04744. A protocol for the production of a
transgenic rat can be found in Bader and Ganten, Clinical and
Experimental Pharmacology and Physiology, Supp. 3:S81-S87, 1996. A
protocol for the production of a transgenic cow can be found in
Transgenic Animal Technology, A Handbook, 1994, ed., Carl A.
Pinkert, Academic Press, Inc. A protocol for the production of a
transgenic sheep can be found in Transgenic Animal Technology, A
Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc.
Reporter Genes
[0133] Promoter activity can be assayed by coupling a reporter gene
to a promoter of interest (e.g., the HGF, HGFR, integrin .alpha.9
or stanniocalcin 1 promoter). The reporter gene can be any gene
which encodes a detectable product, preferably one which can be
detected with relative ease, e.g., a gene product which is
fluorescent, or which catalyzes a reaction which can be determined
by formation of a colored, fluorescent, or luminescent product. For
example, the reporter gene can encode an enzyme, e.g., an enzyme
which produces a detectable product, e.g., a colored, fluorescent,
luminescent product. Reporter genes are known in the art and
include a .beta.-galactosidase gene, a luciferase gene, a green
fluorescent protein gene, a cyan fluorescent protein, a yellow
fluorescent protein, a red fluorescent protein, an alkaline
phosphatase gene, a horseradish peroxidase gene, a .beta.-lactamase
gene, or a chloramphenicol acetyl transferase gene. Reporter genes
are described in, for example, Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989.
IV. Diagnostic Assays
A. Nucleic Acid and Protein Detection
[0134] Arrays are useful molecular tools for characterizing a
sample, e.g., a sample from a subject. For example, an array having
capture probes for multiple genes, including probes for HGF, HGFR,
integrin .alpha.9 and stanniocalcin 1 nucleic acids, or for
multiple proteins. Arrays can have many addresses, e.g., locatable
sites, on a substrate. The featured arrays can be configured in a
variety of formats, non-limiting examples of which are described
below.
[0135] The substrate can be opaque, translucent, or transparent.
The addresses can be distributed, on the substrate in one
dimension, e.g., a linear array; in two dimensions, e.g., a planar
array; or in three dimensions, e.g., a three dimensional array. The
solid substrate may be of any convenient shape or form, e.g.,
square, rectangular, ovoid, or circular.
[0136] Arrays can be fabricated by a variety of methods, e.g.,
photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;
5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow
methods as described in U.S. Pat. No. 5,384,261), pin based methods
(e.g., as described in U.S. Pat. No. 5,288,514), and bead based
techniques (e.g., as described in PCT US/93/04145).
[0137] The capture probe can be a single-stranded nucleic acid, a
double-stranded nucleic acid (e.g., which is denatured prior to or
during hybridization), or a nucleic acid having a single-stranded
region and a double-stranded region. Preferably, the capture probe
is single-stranded. The capture probe can be selected by a variety
of criteria, and preferably is designed by a computer program with
optimization parameters. The capture probe can be selected to
hybridize to a sequence rich (e.g., non-homopolymeric) region of
the gene. The T.sub.m of the capture probe can be optimized by
prudent selection of the complementarity region and length.
Ideally, the T.sub.m of all capture probes on the array is similar,
e.g., within 20, 10, 5, 3, or 2.degree. C. of one another.
[0138] The isolated nucleic acid is preferably mRNA that can be
isolated by routine methods, e.g., including DNase treatment to
remove genomic DNA and hybridization to an oligo-dT coupled solid
substrate (e.g., as described in Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y). The substrate is washed, and
the mRNA is eluted.
[0139] The isolated mRNA can be reversed transcribed and optionally
amplified, e.g., by reverse transcription polymerase chain reaction
(RT-PCR), e.g., as described in (U.S. Pat. No. 4,683,202). The
nucleic acid can be an amplification product, e.g., from PCR (U.S.
Pat. Nos. 4,683,196 and 4,683,202); rolling circle amplification
("RCA," U.S. Pat. No. 5,714,320), isothermal RNA amplification or
NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517), and
strand displacement amplification (U.S. Pat. No. 5,455,166). The
nucleic acid can be labeled during amplification, e.g., by the
incorporation of a labeled nucleotide. Examples of preferred labels
include fluorescent labels, e.g., red-fluorescent dye Cy5.TM.
(Amersham) or green-fluorescent dye Cy3.TM. (Amersham), and
chemiluminescent labels, e.g., as described in U.S. Pat. No.
4,277,437. Alternatively, the nucleic acid can be labeled with
biotin, and detected after hybridization with labeled streptavidin,
e.g., streptavidin-phycoerythrin (Molecular Probes).
[0140] The labeled nucleic acid can be contacted to the array. In
addition, a control nucleic acid or a reference nucleic acid can be
contacted to the same array. The control nucleic acid or reference
nucleic acid can be labeled with a label other than the sample
nucleic acid, e.g., one with a different emission maximum. Labeled
nucleic acids can be contacted to an array under hybridization
conditions. The array can be washed, and then imaged to detect
fluorescence at each address of the array.
[0141] The expression level of a HGF or HGFR protein can be
determined using an antibody specific for the polypeptide (e.g.,
using a western blot or an ELISA assay). Moreover, the expression
levels of multiple proteins, including HGF and HGFR, can be rapidly
determined in parallel using a polypeptide array having antibody
capture probes for each of the polypeptides. Antibodies specific
for a polypeptide can be generated by a method described herein
(see "Antibody Generation").
[0142] A low-density (96 well format) protein array has been
developed in which proteins are spotted onto a nitrocellulose
membrane (Ge (2000) Nucleic Acids Res. 28, e3, I-VII). A
high-density protein array (e.g., 100,000 samples within
222.times.222 mm) used for antibody screening can be produced by
spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et
al. (1999) Anal. Biochem. 270:103-111). See also, e.g., Mendoza et
al. (1999). Biotechniques 27:778-788; MacBeath and Schreiber (2000)
Science 289:1760-1763; and De Wildt et al. (2000). Nature Biotech.
18:989-994. These art-known methods and others can be used to
generate an array of antibodies for detecting the abundance of
polypeptides in a sample. The sample can be labeled, e.g.,
biotinylated, for subsequent detection with streptavidin coupled to
a fluorescent label. The array can then be scanned to measure
binding at each address.
[0143] The nucleic acid and polypeptide arrays of the invention can
be used in wide variety of applications. For example, the arrays
can be used to analyze a patient sample. The sample is compared to
data obtained previously, e.g., known clinical specimens or other
patient samples. Further, the arrays can be used to characterize a
cell culture sample, e.g., to determine a cellular state after
varying a parameter, e.g., exposing the cell culture to an antigen,
a transgene, or a test compound.
[0144] The expression data can be stored in a database, e.g., a
relational database such as a SQL database (e.g., Oracle or Sybase
database environments). The database can have multiple tables. For
example, raw expression data can be stored in one table, wherein
each column corresponds to a gene being assayed, e.g., an address
or an array, and each row corresponds to a sample. A separate table
can store identifiers and sample information, e.g., the batch
number of the array used, date, and other quality control
information.
[0145] Expression profiles obtained from gene expression analysis
on an array can be used to compare samples and/or cells in a
variety of states as described in Golub et al. ((1999) Science
286:531). In one embodiment, expression (e.g., mRNA expression or
protein expression) information for a gene encoding HGF and/or a
gene encoding HGFR are evaluated, e.g., by comparison a reference
value, e.g., a reference value. Reference values can be obtained
from a control, a reference subject. Reference values can also be
obtained from statistical analysis, e.g., to provide a reference
value for a cohort of subject, e.g., age and gender matched
subject, e.g., normal subjects or subject who have or at risk for a
lymphatic or lymphatic system-related disorder. Statistical
similarity to a particular reference (e.g., to a reference for a
risk-associated cohort) or a normal cohort can be used to provide
an assessment (e.g., an indication of risk a lymphatic disorder) to
a subject, e.g., a subject who has not a prior lymphatic disorder,
a subject who has a risk for a lymphatic disorder (e.g., a genetic
predisposition), or a subject who has had a lymphatic disorder.
[0146] Subjects suitable for treatment can also be evaluated for
expression and/or activity of HGF/HGFR pathway activity, e.g., HGF
expression, HGFR expression, integrin .alpha.9 expression and
stanniocalcin 1 expression, as well as modification states or other
parameters associated with these factors. In some embodiments,
subjects can be identified as suitable for treatment if the
expression and/or activity for HGF and/or HGFR is altered (e.g.,
elevated) relative to a reference, e.g., reference value, e.g., a
reference value associated with normal.
[0147] Subjects who are being administered an agent described
herein or other treatment for a lymphatic or lymphatic
system-related disorder can be evaluated as described for
expression and/or activity of HGF and/or HGFR. The subject can be
evaluated at multiple times. e.g., at multiple times during a
course of therapy, e.g., during a therapeutic regimen. Treatment of
the subject can be modified depending on how the subject is
responding to the therapy. For example, a reduction in HGF and/or
HGFR expression or activity can be indicative of responsiveness if
the agent being administered is an antagonist.
[0148] Particular effects mediated by an agent may show a
difference (e.g., relative to an untreated subject, control
subject, or other reference) that is statistically significant
(e.g., P value <0.05 or 0.02). Statistical significance can be
determined by any art known method. Exemplary statistical tests
include: the Students T-test, Mann Whitney U non-parametric test,
and Wilcoxon non-parametric statistical test. Some statistically
significant relationships have a P value of less than 0.05 or
0.02.
B. Methods of Evaluating Genetic Material
[0149] There are numerous methods for evaluating genetic material
to provide genetic information. These methods can be used to
evaluate a genetic locus that includes a gene encoding HGF or a
gene encoding HGFR, as well as other loci. The methods can be used
to evaluate one or more nucleotides, e.g., a coding or non-coding
region of the gene, e.g., in a regulatory region (e.g., a promoter,
a region encoding an untranslated region or intron, and so
forth).
[0150] Nucleic acid samples can analyzed using biophysical
techniques (e.g., hybridization, electrophoresis, and so forth),
sequencing, enzyme-based techniques, and combinations-thereof. For
example, hybridization of sample nucleic acids to nucleic acid
microarrays can be used to evaluate sequences in an mRNA population
and to evaluate genetic polymorphisms. Other hybridization based
techniques include sequence specific primer binding (e.g., PCR or
LCR); Southern analysis of DNA, e.g., genomic DNA; Northern
analysis of RNA, e.g., mRNA; fluorescent probe based techniques
(see, e.g., Beaudet et al. (2001) Genome Res. 11 (4):600-8); and
allele specific amplification. Enzymatic techniques include
restriction enzyme digestion; sequencing; and single base extension
(SBE). These and other techniques are well known to those skilled
in the art.
[0151] Electrophoretic techniques include capillary electrophoresis
and Single-Strand Conformation Polymorphism (SSCP) detection (see,
e.g., Myers et al. (1985) Nature 313:495-8 and Ganguly (2002) Hum
Mutat. 19(4):334-42). Other biophysical methods include denaturing
high pressure liquid chromatography (DHPLC).
[0152] In one embodiment, allele specific amplification technology
that depends on selective PCR amplification may be used to obtain
genetic information. Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the center of
the molecule (so that amplification depends on differential
hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448)
or at the extreme 3' end of one primer where, under appropriate
conditions, mismatch can prevent, or reduce polymerase extension
(Prossner (1993) Tibtech 11:238). In addition, it is possible to
introduce a restriction site in the region of the mutation to
create cleavage-based detection (Gasparini et al. (1992) Mol. Cell
Probes 6:1). In another embodiment, amplification can be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0153] Enzymatic methods for detecting sequences include
amplification based-methods such as the polymerase chain reaction
(PCR; Saiki, et al. (1985) Science 230:1350-1354) and ligase chain
reaction (LCR; Wu. et al. (1989) Genomics 4:560-569; Barringer et
al. (1990), Gene 1989:117-122; F. Barany (1991) Proc. Natl. Acad.
Sci. USA 1988:189-193); transcription-based methods utilize RNA
synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat.
Nos. 6,066,457; 6,132,997; and 5,716,785; Sarkar et al., (1989)
Science 244:331-34; Stofler et al., (1988) Science 239:491); NASBA
(U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517); rolling
circle amplification (RCA; U.S. Pat. Nos. 5,854,033 and 6,143,495)
and strand displacement amplification (SDA; U.S. Pat. Nos.
5,455,166 and 5,624,825). Amplification methods can be used in
combination with other techniques.
[0154] Other enzymatic techniques include sequencing using
polymerases, e.g., DNA polymerases and variations thereof such as
single base extension technology. See, e.g., U.S. Pat. Nos.
6,294,336; 6,013,431; and 5,952,174.
[0155] Fluorescence based detection can also be used to detect
nucleic acid polymorphisms. For example, different terminator
ddNTPs can be labeled with different fluorescent dyes. A primer can
be annealed near or immediately adjacent to a polymorphism, and the
nucleotide at the polymorphic site can be detected by the type
(e.g., "color") of the fluorescent dye that is incorporated.
[0156] Hybridization to microarrays can also be used to detect
polymorphisms, including SNPs. For example, a set of different
oligonucleotides, with the polymorphic nucleotide at varying
positions with the oligonucleotides can be positioned on a nucleic
acid array. The extent of hybridization as a function of position
and hybridization to oligonucleotides specific for the other allele
can be used to determine whether a particular polymorphism is
present. See, e.g., U.S. Pat. No. 6,066,454.
[0157] In one implementation, hybridization probes can include one
or more additional mismatches to destabilize duplex formation and
sensitize the assay. The mismatch may be directly adjacent to the
query position, or within 10, 7, 5, 4, 3, or 2 nucleotides of the
query position. Hybridization probes can also be selected to have a
particular T.sub.m, e.g., between 45-60.degree. C., 55-65.degree.
C., or 60-75.degree. C. In a multiplex assay, T.sub.ms can be
selected to be within 5, 3, or 2.degree. C. of each other.
[0158] It is also possible to directly sequence the nucleic acid
for a particular genetic locus, e.g., by amplification and
sequencing, or amplification, cloning and sequence. High throughput
automated (e.g., capillary or microchip based) sequencing apparati
can be used. In still other embodiments, the sequence of a protein
of interest is analyzed to infer its genetic sequence. Methods of
analyzing a protein sequence include protein sequencing, mass
spectroscopy, sequence/epitope specific immunoglobulins, and
protease digestion.
[0159] Any combination of the above methods can also be used. The
above methods can be used to evaluate any genetic locus, e.g., in a
method for analyzing genetic information from particular groups of
individuals or in a method for analyzing a polymorphism associated
with a lymphatic or lymphatic system-related disorder, e.g., in a
gene encoding HGF, HGFR, integrin .alpha.9 or stanniocalcin 1.
C. In Vivo Imaging
[0160] HGFR HGF/HGFR binding agents (e.g., antibodies) can be used
detecting the presence of HGF and/or HGFR in vivo (e.g., in vivo
imaging in a subject), respectively. The method can be used to
evaluate (e.g., diagnose, localize, or stage) a condition described
herein, e.g., a lymphatic disorder or risk for such a disorder. The
method includes: (i) administering to a subject (and optionally a
control subject) a HGF/HGFR binding agent (e.g., an antibody that
binds to HGF or HGFR), under conditions that allow interaction of
the binding agent and HGF or HGFR to occur; and (ii) detecting the
binding agent, for example, to locate or otherwise identify HGF or
HGFR expressing cells. A statistically significant increase in the
amount of the complex in the subject relative to the reference,
e.g., the control subject or subject's baseline, can be a factor
that may lead to a diagnosis of a lymphatic or lymphatic
system-related disorder or risk for such a disorder.
[0161] Preferably, the HGF/HGFR binding agent used in the in vivo
(and also in vitro) diagnostic methods is directly or indirectly
labeled with a detectable substance to facilitate detection of the
bound or unbound binding agent. Suitable detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. In one embodiment,
the HGF or HGFR binding protein is coupled to a radioactive ion,
e.g., indium (111In), iodine (131I or 125I), yttrium (90Y),
actinium (225Ac), bismuth (212Bi or 213Bi), sulfur (35S), carbon
(14C), tritium (3H), rhodium (188Rh), or phosphorous (32P). In
another embodiment, the HGF/HGFR binding protein is labeled with an
NMR contrast agent.
[0162] In one aspect, the invention features a method of imaging
vasculature (e.g., lymphatic vasculature) in a patient who is at
risk for a lymphatic or lymphatic system-related disorder, or has
such a disorder which is progressing. The method includes:
providing an agent that binds to HGF or HGFR, e.g., an agent
described herein, wherein the protein is physically associated to
an imaging agent; administering the agent to a patient, e.g., with
a risk for a lymphatic or lymphatic system-related disorder; and
locating the agent within the patient, e.g., by imaging the
patient, e.g., to detect HGF or HGFR expressing cells.
EXAMPLES
Example 1
HGFR is Expressed at a Higher Level in Lymphatic Endothelial Cells
than in Blood Vascular Endothelial Cells and is Functional
[0163] Quantitative real-time RT-PCR (QPCR) confirmed that three
independently established lines of primary LEC expressed high
levels of mRNA of the major lymphatic lineage markers Prox1 and
LYVE-1 but expressed low levels of the blood vascular lineage
marker Flt-1, relative to blood vascular endothelial cells (BVEC)
(FIG. 1A-C). HGFR mRNA levels in LEC were measured by QPCR and
found to be at more than 2-fold higher than levels in BVEC (FIG.
1D). In addition, immunoprecipitation and western blot analyses
demonstrated that HGFR protein expression was higher in LEC than in
BVEC (FIG. 1E). Since treatment of LEC with 30 ng/ml HGF resulted
in increased phosphorylation of HGFR (FIG. 1F), HGFR is functional
in LEC. In sum, HGFR is expressed at a higher level in LEC than in
BVEC and is activated when LEC are treated with HGF.
Example 2
HGFR Expression is Expressed in Lymphatic Vessels During
Inflammation and Tissue Repair in Vivo
[0164] Differential immunofluorescence analyses of normal mouse
skin were performed, using antibodies against HGFR and against the
lymphatic-specific hyaluronan receptor LYVE-1. Little or no
expression of HGFR was detected in quiescent lymphatic vessels in
normal skin. In order to determine whether HGFR might be
upregulated by lymphatic endothelium during pathological processes,
we immunostained samples of chronically inflamed murine skin
obtained from experimentally induced delayed-type hypersensitivity
reactions in VEGF-A transgenic (VEGF-TG) mice that are
characterized by lymphatic vessel enlargement and proliferation,
Kunstfeld et al. (2004), Blood, 104(4):1048-1057. Seven days after
induction of skin inflammation, enlarged LYVE-1-positive lymphatic
vessels were detected in VEGF-TG mice but not in wild-type mice
(FIG. 2A, D). LYVE-1-positive lymphatic vessels strongly expressed
HGFR, whereas little or no HGFR expression was detected in the
normal lymphatic vessels of wild-type mice (FIG. 2A-F).
[0165] Two to three weeks after experimentally-induced
full-thickness skin wounds in mice, pronounced lymphangiogenesis is
found within the granulation tissue. Double immunofluorescence
analyses of wound tissue at day 21 after wounding revealed several
LYVE-1-positive lymphatic vessels that also expressed HGFR (FIG.
2G-L).
[0166] To further characterize the possible role of HGFR during
embryonic lymphatic vessel formation, mouse embryonic tissues were
examined at embryonic days (E) 10.5 to 14.5 when active budding and
proliferation of lymphatic vessel progenitors occurs. At E11.5,
LYVE-1 expression was detected on endothelial cells of the anterior
cardinal vein. These cells expressed little or no HGFR, whereas
HGFR expression was already detected within the pharyngeal region
of the foregut and in mesenchymal cells (FIG. 3A-C). However, at
E12.5, HGFR expression was clearly detectable on LYVE-1-positive
endothelial cells of the anterior cardinal vein (FIG. 3F:
arrowheads), whereas only occasional HGFR expression was found on
endothelial cells lining the primitive lymph sacs, overlapping with
LYVE-1 reactivity (FIG. 3D-F). By E14.5, strong HGFR expression was
detected on the vast majority of LYVE-1-positive lymphatic
endothelial cells (FIG. 3G-I). At this stage, HGFR was still weakly
expressed by endothelial cells lining the jugular vein and artery,
which were LYVE-1-negative.
Example 3
HGF Directly Promotes LEC Proliferation and Migration
[0167] HGF is the only known ligand of HGFR and has been shown to
induce proliferation and migration of human vascular endothelial
cells (HVEC), e.g., Bussolino et al. (1992), J. Cell Biol.,
119:629-641. To investigate whether the differential expression
levels of HGFR by LEC versus BVEC might result in their
differential response toward HGF stimulation, we next investigated
the effects of HGF on LEC versus BVEC proliferation in vitro. HGF
potently induced LEC proliferation with a minimal effective
concentration of 1 ng/ml (p<0.01), as compared with untreated
control cultures. Although HGF also induced BVEC proliferation at
this concentration, the extent of growth stimulation was higher in
LEC than in BVEC (FIG. 4A). Thus far, VEGF-C and VEGF-D are the
only known growth factors that directly promote LEC proliferation
via activation of the VEGF receptor-3 (VEGFR-3), e.g., Jussila and
Alitalo (2002), Physiol Rev, 82:673-700, and effects of FGF-2 on
lymphangiogenesis have been proposed to be the result of
upregulation of VEGFR-3 ligands because they could be inhibited by
blockade of the VEGFR-3 pathway, e.g., Chang et al. (2004), PNAS,
101:11658-11663; Kubo et al. (2002), PNAS, 99:8868-8873. To
investigate whether HGF directly or indirectly stimulates LEC
proliferation, we next treated LEC with HGF, in the presence or
absence of blocking antibodies against VEGFR-3 or HGFR. Incubation
of LEC with a HGFR blocking antibody potently blocked the
stimulation of LEC proliferation by HGF (p<0.001), whereas
incubation with a VEGFR-3 blocking antibody--at a dose that
efficiently blocked growth stimulation by VEGF-C (data not
shown)--or with control IgG did not affect HGF-induced
proliferation (FIG. 4B). These results indicate that HGF-induced
LEC proliferation occurs independently from activation of the
VEGF-R3 pathway and is dependent upon efficient binding of HGF to
its receptor.
[0168] HGF treatment also dose-dependently promoted migration of
LEC and BVEC, with a minimal effective concentration of 3 ng/ml
(FIG. 4C). To investigate whether HGF stimulation might also
promote the formation of lymphatic tubes in vitro, confluent LEC
cultures were overlaid with type I collagen as previously described
Hirakawa et al. (2003), Am. J. Pathol., 162:575-586. HGF potently
induced cord formation by LEC with a minimal effective dose of 3
ng/ml (p<0.001), as compared with untreated control cultures
(FIG. 5A, B).
Example 4
HGF Promotes Lymphatic Vessel Formation in Vivo
[0169] To investigate whether HGF might also induce
lymphangiogenesis in vivo, we implanted matrigels with or without
HGF subcutaneously into FVB mice as described in Hirakawa et al.
(2003), Am. J. Pathol., 162:575-586. Immunostaining for the
lymphatic-specific glycoprotein podoplanin, e.g., Schacht et al.
(2003), EMBO J., 22:3546-3556, revealed pronounced formation of new
lymphatic vessels within HGF-containing matrigels at day 7 after
implantation, whereas no lymphatic vessels were observed within
control matrigels (FIG. 5C,D).
Example 5
Systemic Blockade of HGFR Inhibits Lymphatic Vessel Enlargement
During Experimental Skin Inflammation
[0170] Because we found that HGFR was strongly expressed by the
enlarged lymphatic vessels during experimental skin inflammation in
mice, we next investigated whether HGF might directly contribute to
lymphatic vessel enlargement in vivo. Delayed-type hypersensitivity
reactions were induced by topical application to mouse ears as
described Kunstfeld et al. (2004), Blood, 104(4):1048-1057. One day
prior to induction of experimental inflammation, 100 .mu.g of a
blocking antibody against HGFR or an equal amount of control
immunoglobulin G were injected intraperitoneally.
Immunofluorescence analyses at 24 hours after induction of
inflammation revealed a greatly reduced size of lymphatic vessels
in mice that had received treatment with the HGF-R blocking
antibody, as compared with mice that had received control IgG (FIG.
6A, B). Computer-assisted morphometric analyses of sections stained
for LYVE-1 and CD31 demonstrated that the average size of lymphatic
vessels, and the percentage of tissue area covered by lymphatic
vessels, were significantly decreased after injection of the HGF-R
blocking antibody (P<0.01), as compared with control IgG-treated
mice (FIG. 6C, D). The density of lymphatic vessels was not
significantly different between the two treatment groups (FIG.
6E).
Example 6
HGF Promotes LEC Migration via Integrin .alpha.9
[0171] To define possible molecular mechanisms that might mediate
the effects of HGF on LEC, two independent lines of LEC were
incubated with or without 30 ng/ml HGF for 6 hours, followed by
microarray analyses using the Affymetrix HU133v2.TM. arrays. We
found that stanniocalcin 1 was one of the most highly up-regulated
genes after HGF treatment. Moreover, the expression of the integrin
alpha 9 was also significantly upregulated after HGF treatment.
These results were confirmed by QPCR analysis (FIG. 7A).
[0172] Co-incubation of LEC with HGF in the presence or absence of
a specific integrin alpha 9 blocking antibody revealed that
blockade of the integrin alpha 9 partially blocked HGF-induced
migration (p=0.0143), whereas incubation with a HGF-R blocking
antibody completely inhibited the effect of HGF on LEC migration
(p=0.0074) (FIG. 7B).
Example 7
HGF Promotes Lymphatic Vessel Formation in Vivo Independently of
VEGF
[0173] To determine the effect of HGF overexpression in vivo, the
lymphatic vasculature was investigated in previously established
metallothionein I promoter-driven HGF transgenic mice (Takayama et
al. (1996) Proc. Natl. Acad. Sci. USA, 93:5866-5871). The analysis
focused on the skin and the small intestine where lymphatic vessels
are most abundant and where abnormalities of the lymphatic system
are most easily detected in genetic mouse models. Vascular
enlargement was detected in the mucosa and submucosa of the ileum
in HGF transgenic mice (FIG. 8B), as compared with wild-type mice
(FIG. 8A). Immunofluorescence stains for the lymphatic-specific
marker podoplanin revealed pronounced dilation of central lacteals
and enlargement of lymphatic vessels in the submucosa of the ileum
in HGF transgenic mice (FIG. 8C, D). Podoplanin stains also
revealed an increased number and an enlargement of lymphatic
vessels in the skin of HGF transgenic mice (FIG. 8G, H), whereas no
major histological abnormalities were observed (FIG. 8E, F).
Enhanced lymphatic vessel formation and enlargement were also
observed in the duodenum and liver of HGF transgenic mice.
[0174] To examine whether HGF promotes the formation of new
lymphatic vessels directly or indirectly via the VEGFR-3 pathway,
slow-release pellets (with or without HGF) were implanted
subcutaneously into mouse ears, and mice were treated systemically
with a blocking antibody against mouse VEGFR-3 or with control IgG.
After 14 days, immunofluorescence stains for CD31 and LYVE-1
revealed pronounced lymphatic vessel formation surrounding
HGF-containing pellets, but not surrounding control pellets (FIG.
9A-C). However, treatment with an anti-VEGFR-3 blocking antibody
did not prevent lymphatic vessel formation induced by HGF (FIG. 9B,
C). Mice implanted with HGF-containing pellets showed moderately
enhanced formation of blood vessels.
[0175] The results shown in these examples indicate that HGF is a
protein target for controlling lymphangiogenesis.
OTHER EMBODIMENTS
[0176] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. For example, combinations of nucleic acid
and antibody modulators of HGF and HGFR activity are contemplated.
Accordingly, other embodiments are within the scope of the
following claims.
* * * * *