U.S. patent application number 15/310514 was filed with the patent office on 2017-03-16 for compositions comprising il-31 and uses thereof.
The applicant listed for this patent is RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL SCIENCES. Invention is credited to Ami ARONHEIM, Ella FREMDER, Yuval SHAKED.
Application Number | 20170072019 15/310514 |
Document ID | / |
Family ID | 54479403 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170072019 |
Kind Code |
A1 |
FREMDER; Ella ; et
al. |
March 16, 2017 |
COMPOSITIONS COMPRISING IL-31 AND USES THEREOF
Abstract
A method for treating cancer and/or preventing or reducing
metastasis or treating angiogenesis related disorders comprising as
the step of administering IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No.
1.
Inventors: |
FREMDER; Ella; (Haifa,
IL) ; ARONHEIM; Ami; (Binyamina, IL) ; SHAKED;
Yuval; (Binyamina, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL
SCIENCES |
Haifa |
|
IL |
|
|
Family ID: |
54479403 |
Appl. No.: |
15/310514 |
Filed: |
May 12, 2015 |
PCT Filed: |
May 12, 2015 |
PCT NO: |
PCT/IL2015/050498 |
371 Date: |
November 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61991641 |
May 12, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/33 20130101;
C07K 14/54 20130101; C12N 15/113 20130101; C12N 15/1136 20130101;
A61K 38/20 20130101; C07K 2319/30 20130101; A61K 2039/55527
20130101; C07K 2319/50 20130101; A61K 9/1273 20130101; A61K 39/39
20130101; C12N 2310/531 20130101; C12N 2310/14 20130101 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 9/127 20060101 A61K009/127; C07K 14/54 20060101
C07K014/54 |
Claims
1. A method for treating cancer and/or preventing or reducing
metastasis comprising the step of administering IL-31 or peptide
which is at least about 70%, homologous to the IL-31 sequence as
set forth in SEQ ID No. 1, a fused protein comprising IL-31 or
peptide which is at least about 70%, homologous to the IL-31
sequence as set forth in SEQ ID No. 1, an agent which up-regulates
IL-31, an IL-31 receptor agonist or a complex comprising either
IL-31 or peptide which is at least about 70%, homologous to the
IL-31 sequence as set forth in SEQ ID No. 1 or a fused protein that
comprises IL-31 or peptide which is at least about 70%, homologous
to the IL-31 sequence as set forth in SEQ ID No. 1, to a subject in
need, thereby treating cancer and/or reducing or preventing
metastasis.
2. A method for treating angiogenesis related disorder comprising
the step of administering IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No. 1,
a fused protein comprising IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No. 1,
an agent which up-regulates IL-31, an IL-31 receptor agonist or a
complex comprising either IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No. 1
or a fused protein that comprises IL-31 or peptide which is at
least about 70%, homologous to the IL-31 sequence as set forth in
SEQ ID No. 1 to a subject in need, thereby treating the
angiogenesis related disorder.
3. A fused protein comprising IL-31 or peptide which is at least
about 70%, homologous to the IL-31 sequence as set forth in SEQ ID
No. 1.
4. The fused protein of claim 3, wherein said IL-31 to the IL-31
sequence as set forth in SEQ ID No. 1 is attached to a heterologous
amino acid sequence.
5. The fused protein of claim 4, wherein said heterologous amino
acid sequence comprises an immunoglobulin amino acid sequence.
6. The fused protein of claim 5, wherein the immunoglobulin amino
acid sequence comprises IgG.
7. The fused protein of claim 3, wherein the fused protein further
comprises a cleavage site for an enzyme.
8. The fused protein of claim 7, wherein the enzyme is trypsin,
PSA, MMP-9/2 or cathepsin or any combination thereof.
9-14. (canceled)
15. A complex comprising IL-31 or a fused protein comprising IL-31
or peptide which is at least about 70%, homologous to the IL-31
sequence as set forth in SEQ ID No. 1 and non-proteinaceous or
proteinaceous moiety.
16. The complex of claim 15, wherein the non proteinaceous is
polyethylene glycol (PEG) or derivative thereof, polyvinyl
pyrrolidone (PVP), divinyl ether, albumin, maleic anhydride
copolymer (DIVEMA), polysialic acid (PSA), poly(styrene comaleic
anhydride) (SMA), hyaluronic acid (HA), alginic acid (AA),
polyhydroxyethyl methacrylate (Poly-HEMA), glyme or
polyisopropylacrylamide or any combination thereof.
17. The complex of claim 15, in a form of a liposome or a
micelle.
18-19. (canceled)
20. The method of claim 1, wherein the cancer is selected from the
group consisting of brain cancer, oropharyngeal cancer,
nasopharyngeal cancer, renal cancer, biliary cancer, prostatic
cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni
tumors, thyroid cancer, parathyroid cancer, pituitary tumors,
adrenal gland tumors, osteogenic sarcoma tumors, multiple
neuroendrcine type I and type II tumors, breast cancer, lung
cancer, head & neck cancer, prostate cancer, esophageal cancer,
tracheal cancer, skin cancer brain cancer, liver cancer, bladder
cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine
cancer, cervical cancer, testicular cancer, colon cancer, rectal
cancer and skin cancer.
21. The method of claim 2, wherein the related disorder is selected
from the group consisting of cancer, arthritis, rheumatoid
arthritis, atherosclerotic plaques, conical graft
neovascularization, hypertrophic or keloid scars, proliferative
retinopathy, diabetic retinopathy, macular degeneration, age
related macular degeneration (AMD), granulation, neovascular
glaucoma, uveitis, liver fibrosis, lung fibrosis asthma, Idiopathic
Pulmonary Fibrosis (IPF), Myelofibrosis and Primary Sclerosing
Cholangitis.
22. The method claim 1, wherein the cancer is hematological
malignance selected from the group consisting of multiple myeloma,
acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML),
chronic lymphoblasic leukemia, chronic myeloid leukemia (CML) or
mesothieloma.
Description
BACKGROUND OF THE INVENTION
[0001] Cancer is a leading cause of death in most countries, and
the result of billions of dollars in healthcare expense around the
world. It is now well established that a variety of cancers are
caused, at least in part, by genetic abnormalities that result in
either the overexpression of cancer causing genes, called
"oncogenes," or from loss of function mutations in protective
genes, often called "tumor suppressor" genes. One of the main
obstacles in clinical oncology is that tumors can usually resist
therapy, leading to tumor re-growth and even metastasis. There are
many reasons that may explain why tumor cells become resistant to
anti-cancer drugs, such as the ability of tumor cells to undergo
selection for acquired resistance.
[0002] It has been demonstrated that several types of bone marrow
derived cells home-in on chemotherapy-treated tumors and colonize
there, leading to increased angiogenesis and metastasis.
[0003] The contribution of host cells to tumor growth is not solely
dependent on angiogenesis. Recent studies indicated that immune
cells, such as macrophages, also related to as tumor associated
macrophages (TAMs) contribute to tumor growth. Macrophages are
myeloid cells that are linked with inflammation. There are two main
phenotypes of macrophages: M1 and M2. These two phenotypes are
associated not only with tumors, but also with other pathological
and physiological conditions related to the inflammatory cascade.
During the inflammatory process M1 macrophages initially arrive and
colonize the damaged tissue. They secrete various cytokines and
chemokines at the inflammatory site, which ignite the inflammatory
cascade. M1 macrophages have high phagocytotic properties and they
secrete pro-inflammatory factors. On the other hand, M2 macrophages
colonize the inflammatory tissue only a few days after M1
macrophages colonized the tissue. Their role is to stop the
inflammatory process, and to initiate a regeneration process.
Therefore, they secrete anti-inflammatory cytokines and growth
factors known to repair damaged tissue, among those are factors
promoting cell proliferation, migration, and activation. In cancer,
M2 macrophages were found to substantially contribute to the
tumorigenesis process and to metastasis, while M1 macrophages most
likely contribute to the inhibition of pro-tumorigenic properties
of cancer cells by creating an acute inflammatory process.
[0004] IL-31 is an immunoregulatory cytokine that is mainly
produced by activated Th2 cells. IL-31 acts through the
heterodimeric receptors of IL-31 (IL-31R) and oncostatin M receptor
(OSMR), which are expressed on IL-31 activated monocytes and on
epithelial cells.
[0005] The possible role of IL-31 as an anti-cancer compound was
not investigated as of to date.
[0006] There is a need in identifying a new treatment for
cancer.
SUMMARY OF THE INVENTION
[0007] This application is directed to a method for treating cancer
and/or preventing or reducing metastasis comprising the step of
administering IL-31, a fused protein comprising IL-31, an agent
which up-regulates IL-31, an IL-31 receptor agonist or a complex
comprising either IL-31 or a fused protein that comprises IL-31 to
a subject in need, thereby treating cancer and/or reducing or
preventing metastasis.
[0008] In one embodiment of the invention, the cancer is selected
from the group consisting of brain cancer, oropharyngeal cancer,
nasopharyngeal cancer, renal cancer, biliary cancer, prostatic
cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni
tumors, thyroid cancer, parathyroid cancer, pituitary tumors,
adrenal gland tumors, osteogenic sarcoma tumors, multiple
neuroendocrine type I and type II tumors, breast cancer, lung
cancer, head & neck cancer, prostate cancer, esophageal cancer,
tracheal cancer, skin cancer, such as without being limited,
melanoma or squamous cell carcinoma, brain cancer, such as without
being limited, neuroblastoma, glioblastoma, astrositoma, liver
cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian
cancer, uterine cancer, cervical cancer, testicular cancer, colon
cancer, rectal cancer or skin cancer. In some embodiments, the
cancer is hematological malignancies, such as, multiple myeloma,
acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML),
chronic lymphoblasic leukemia or chronic myeloid leukemia (CML). In
some embodiments, the cancer is mesothieloma.
[0009] This application is further directed to a method for
treating angiogenesis related disorder comprising the step of
administering IL-31, a fused protein comprising IL-31, or a complex
comprising either IL-31 or a fused protein that comprises IL-31 to
a subject in need, thereby treating the angiogenesis related
disorder.
[0010] In one embodiment of the invention, the related disorder is
selected from the group consisting of cancer, arthritis, rheumatoid
arthritis, atherosclerotic plaques, corneal graft
neovascularization, hypertrophic or keloid scars, proliferative
retinopathy, diabetic retinopathy, macular degeneration or age
related macular degeneration (AMD), granulation, neovascular
glaucoma and uveitis. In some embodiments, the angiogenesis related
disorder is "fibrosis-related diseases, e.g., liver fibrosis and
lung fibrosis. In some embodiments, the angiogenesis related
disorder is asthma.
[0011] This application is further directed to a fused protein
comprising IL-31. In some embodiments, the IL-31 is attached to a
heterologous amino acid sequence. In some embodiments, the
heterologous amino acid sequence comprises an immunoglobulin amino
acid sequence. In some embodiments, the immunoglobulin amino acid
sequence comprises IgG.
[0012] In one embodiment of the invention, the fused protein
further comprises IgG.
[0013] In one embodiment of the invention, the fused protein
further comprises a cleavage site for an enzyme.
[0014] In one embodiment of the invention, the enzyme is trypsin,
PSA, MMP-9/2 or cathepsin or any combination thereof.
[0015] This application is further directed to a nucleic acid
encoding a fused protein comprising IL-31.
[0016] In one embodiment of the invention, the nucleic acid further
comprises a nucleic acid encoding IgG.
[0017] In one embodiment of the invention, the nucleic acid further
comprises a nucleic acid encoding a cleavage site for enzymes.
[0018] In one embodiment of the invention, the enzyme is trypsin,
PSA, MMP-9/2 or cathepsin or any combination thereof.
[0019] This application is further directed to a vector comprising
the nucleic acid of any one of the previous embodiments.
[0020] This application is further directed to a cell transformed
with a vector comprising the nucleic acid of any one of the
previous embodiments.
[0021] This application is further directed to a complex comprising
IL-31 or a fused protein comprising IL-3 and non-proteinaceous or
proteinaceous moiety.
[0022] In one embodiment of the invention, the non-proteinaceous is
polyethylene glycol (PEG) or derivative thereof, polyvinyl
pyrrolidone (PVP), albumin, divinyl ether, maleic anhydride
copolymer (DIVEMA; and poly(styrene comaleic anhydride) (SMA),
hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl
methacrylate (Poly-HEMA), glyme or polyisopropylacrylamide or any
combination thereof.
[0023] In one embodiment of the invention the complex is in a form
of a liposome or a micelle.
[0024] In some embodiments of the invention, there is provided an
IL-31 protein or peptide which is at least about 70%, homologous to
the IL-31 sequence as set forth in SEQ ID No. 1, a fused protein
comprising IL-31 or peptide which is at least about 70%, homologous
to the IL-31 sequence as set forth in SEQ ID No. 1, an agent which
up-regulates IL-31, an IL-31 receptor agonist or a complex
comprising either IL-31 or peptide which is at least about 70%,
homologous to the IL-31 sequence as set forth in SEQ ID No. 1 or a
fused protein that comprises IL-31 or peptide which is at least
about 70%, homologous to the IL-31 sequence as set forth in SEQ ID
No. 1 for use in treating cancer and/or preventing or reducing
metastasis.
[0025] In some embodiments, there is provided an IL-31 protein or
peptide which is at least about 70%, homologous to the IL-31
sequence as set forth in SEQ ID No. 1, a fused protein comprising
IL-31 or peptide which is at least about 70%, homologous to the
IL-31 sequence as set forth in SEQ ID No. 1, an agent which
up-regulates IL-31, an IL-31 receptor agonist or a complex
comprising either IL-31 or peptide which is at least about 70%,
homologous to the IL-31 sequence as set forth in SEQ ID No. 1 or a
fused protein that comprises IL-31 or a peptide which is at least
about 70%, homologous to the IL-31 sequence as set forth in SEQ ID
No. 1 for use in treating angiogenesis related disorders.
[0026] In some embodiments of the invention, there is provided a
method for treating cancer and/or preventing or reducing metastasis
comprising the step of administering IL-31 or peptide which is at
least about 70%, homologous to the IL-31 sequence as set forth in
SEQ ID No. 1, a fused protein comprising IL-31 or peptide which is
at least about 70%, homologous to the IL-31 sequence as set forth
in SEQ ID No. 1, an agent which up-regulates IL-31, an IL-31
receptor agonist or a complex comprising either IL-31 or peptide
which is at least about 70%, homologous to the IL-31 sequence as
set forth in SEQ ID No. 1 or a fused protein that comprises IL-31
or peptide which is at least about 70%, homologous to the IL-31
sequence as set forth in SEQ ID No. 1, to a subject in need,
thereby treating cancer and/or reducing or preventing
metastasis.
[0027] In some embodiments of the invention, there is provided a
method for treating angiogenesis related disorder comprising the
step of administering IL-31 or peptide which is at least about 70%,
homologous to the IL-31 sequence as set forth in SEQ ID No. 1, a
fused protein comprising IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No. 1,
an agent which up-regulates IL-31, an IL-31 receptor agonist or a
complex comprising either IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No. 1
or a fused protein that comprises IL-31 or peptide which is at
least about 70%, homologous to the IL-31 sequence as set forth in
SEQ ID No. 1 to a subject in need, thereby treating the
angiogenesis related disorder.
[0028] In some embodiments of the invention, there is provided a
fused protein comprising IL-31 or peptide which is at least about
70%, homologous to the IL-31 sequence as set forth in SEQ ID No.
1.
[0029] In some embodiments of the invention, there is provided an
IL-31 protein or peptide which is at least about 70%, homologous to
the IL-31 sequence as set forth in SEQ ID No. 1 is attached to a
heterologous amino acid sequence.
[0030] In some embodiments, the heterologous amino acid sequence
comprises an immunoglobulin amino acid sequence, which may be
IgG.
[0031] In some embodiments of the invention, there is provided a
nucleic acid encoding a fused protein comprising IL-31 or peptide
which is at least about 70%, homologous to the IL-31 sequence as
set forth in SEQ ID No. 1.
[0032] In some embodiments, the nucleic acid encodes IgG.
[0033] The fused protein may further comprise a nucleic acid
encoding a cleavage site for enzymes, wherein the enzyme may be
trypsin, PSA, MMP-9/2 or cathepsin or any combination thereof.
[0034] In some embodiments, there is provided a complex comprising
IL-31 or a fused protein comprising IL-31 or peptide which is at
least about 70%, homologous to the IL-31 sequence as set forth in
SEQ ID No. 1 and non-proteinaceous or proteinaceous moiety. The non
proteinaceous may be polyethylene glycol (PEG) or derivative
thereof, polyvinyl pyrrolidone (PVP), divinyl ether, albumin,
maleic anhydride copolymer (DIVEMA), polysialic acid (PSA),
poly(styrene comaleic anhydride) (SMA), hyaluronic acid (HA),
alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), glyme
or polyisopropylacrylamide or any combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The subject matter regarded as the invention is particularly
pointed out and distinctly claimed in the concluding portion of the
specification. The invention, however, both as to organization and
method of operation, together with objects, features, and
advantages thereof, may best be understood by reference to the
following detailed description when read with the accompanying
drawings in which:
[0036] FIGS. 1(A-E): The upper image of FIG. 1A is a western blot
image showing the expression level of IL-31 in various tumor cell
lines and macrophages. Below is a western blot image showing the
level of IL-31 receptor (IL-31R) in various tumor cell lines and
macrophages. A star (*) represents metastatic cells. An underlined
cell type represents a non-metastatic cell, other cell lines are
hematologic cell lines. An inverse correlation between the
expression of IL-31 and its receptor and the metastatic properties
of tumor cells can be seen. FIGS. 1B 1C, 1D and 1E present the
viability of MC38 cells (FIG. 1B), 4T1 cells (FIG. 1C), CT26 cells
(FIG. 1D) and HCT116 cells (FIG. 1E), respectively, in the presence
of escalating doses of recombinant IL-31 (rIL-31) using Alamar-Blue
assay; FIGS. 1D and 1E present the number of cells of MC38 (FIG.
1D) and 4T1 (FIG. 1E) in the presence of different doses of rIL-31
using trypan blue to exclude dead cells;
[0037] FIGS. 2 (A and B): FIG. 2A is a Western blot image comparing
IL-31 expression in lysates of MC38 transfected with shIL-31
plasmid or with scrambled plasmid; FIG. 2B is a graph showing the
percentage of reduction of IL-31 expression in MC38 cells
transfected with shIL-31 plasmid or with scrambled plasmid, as
assessed by densitometry;
[0038] FIGS. 3 (A-C): FIG. 3A compares tumor size (mm.sup.3) in
mice that were injected subcutaneously into the flanks with MC38
cells transfected with either shIL31 or scrambled plasmid; FIG. 3B
presents microvessel density (MVD) and large vessel structures in
tumor removed from mice injected with MC38 cells transfected with
shIL-31 or scrambled plasmids; the number of vessel structures or
cell per field were counted and plotted. FIGS. 3C and 3D present
the number of macrophages (F4/80+ cells) (FIG. 3C) and endothelial
cells (FIG. 3D) in tumor from mice injected with MC38 cells
transfected with shIL-31 or scrambled plasmids;
[0039] FIGS. 4 (A-I) FIG. 4A shows the tumor size (mm.sup.3) in
mice implanted with MC38 cells into the flank and implanted with
minipump containing either rIL-31 or PBS (control); FIG. 4B shows
the tumor size (mm.sup.3) in mice implanted with 4T1 cells to the
mammary fad pad and infused with minipump containing either rIL-31
or PBS (control); FIG. 4C compares microvessel density (MVD) in
tumors that were removed from the mice implanted with MC38 and
infused with either rIL-31 or PBS (control). The tumors were
removed, sectioned and immune-stained with CD 31; nuclear staining
was designated by 4',6-diamidino-2-phenylindole (DAPI); FIG. 4D is
a graph comparing MVD levels in tumors from mice implanted with
MC38 and infused with either 0.7 .mu.g/day rIL-31 or PBS (control).
FIGS. 4E and 4F compare the number of lung metastatic lesions using
H&E staining of lung sections in tumors from mice implanted
with 4T1 cells and infused with either rIL-31 or PBS (control)(FIG.
4E); Arrows represent the metastatic lesions in the lung section.
The quantification of the number of metastatic lesions per field is
provided (FIG. 4F). FIG. 4G shows tumor growth (mm.sup.3) in
NOD-SCID mice that were implanted with HCT116 cells
(2.times.10.sup.6 cells; n=5 mice/group). When the tumors reached a
size of 50 mm.sup.3, the mice were either implanted with pumps
containing 150 .mu.g hIL31-IgG protein or injected ip twice a week
with 50 .mu.g hIL-31-IgG. Tumor growth was assessed over time.
[0040] FIGS. 4H and 4II, the tumors presented in FIG. 4G were
removed at the end point, after the mice were treated with
hIL-31-IgG for two weeks either by pump or by IP injections. (4H)
Tumors were sectioned and stained for CD31 (an endothelial cell
marker). (4I) Quantification of the number of vessels (MVD) per
field is provided.
[0041] FIGS. 5 (A-C): FIG. 5A is a graph comparing the number of M1
macrophage phenotype and M2 macrophage phenotype in J774 cells
cultured in the presence and absence of rIL-31 (100 ng); cells were
immune-stained with F4/80, CD206, and CD11c to evaluate the
percentage of M1 (CD11c+/CD206-) and M2 (CD11c-/CD206+)
macrophages.
[0042] FIG. 5B is a graph comparing the number of M1 macrophage
phenotype and M2 macrophage phenotype in a single cell suspension
from MC38 tumors implanted in C57B16 which, either express IL-31
(ev-scrambled) or not (shIL-31), and that were let to grow until
the endpoint. The percentage of M1 and M2 macrophages colonizing
tumors were analyzed using flow cytometry. FIG. 5C is a graph
comparing the number of M1 macrophage phenotype and M2 macrophage
phenotype in a single cell suspension from MC38 tumors that were
implanted in the flanks of C57Bl/6 mice. When tumors reached
150-200 mm.sup.3, mice were implanted with mini-pumps containing
PBS (control) or recombinant IL-31 in a dose of 0.7 .mu.g/day
(rIL-31). The percentage of M1 and M2 macrophages colonizing tumors
were analyzed using flow cytometry.
[0043] FIG. 6 presents an assessment of MC38 cell viability using
Alamar Blue assay. The cells were cultured in the presence of
escalating doses of mIL-31-IgG. A reduction in cell viability was
observed with the increased concentration of IL-31-IgG.
[0044] FIG. 7 shows an example of staining image for breast cancer
biopsy P-12855/11. High intensity of IL-31R expression is observed
in carcinoma cells of the breast cancer biopsy.
[0045] FIGS. 8 (A-I): shows that IL-31-IgG is stabilized for at
least 72 hours in peripheral blood. 293T cells were transfected
with the IL-31-Ig construct. (8A). Conditioned medium (CM) and
lysates were obtained after 48 hours and were then detected for the
various components of the IL-31 construct by Western Blot. (8B-E).
Detection of the miL-31 part (8B), the mCH2-CH3-IgG part (8C), the
Myc part (8D) and the His part (8E) using the Goat-a-Rat,
G-a-m-IgG, G-a-m-light, G-a-m-light antibodies respectively.
Coomassie Brilliant Blue (CBB) stain of 20 mg purified hIL-31-IgG
and mIL-31-IgG is shown in 8F. (8G-H) The protein IL-31-IgG (both
human and mouse) which was generated and purified has been tested
for its stability in peripheral blood of mice. C57B16 mice were
injected with 30 .mu.g of the indicated IL-31 proteins. Blood was
drawn by retro-orbital sinus at different time points, and plasma
was separated. Plasma (2 .mu.l) was used to detect the various
IL-31 proteins using anti-His-HRP conjugated antibody by Western
Blot. (8G) 30 .mu.g mIL31-IgG Vs. 200 .mu.g mIL31. (8H) 30 .mu.g
hIL31-IgG Vs. 200 .mu.g hIL31.
[0046] FIGS. 9 (A and B): human umbilical vascular endothelial
cells (HUVECs) were seeded in Matrigel-coated 48-well tissue
culture plates (4.times.104 cells/well) and incubated in 20% 1-BS
M-199 medium. Wells were cultured with 100 ng/ml recombinant human
IL-31 or 10 ug/ml human IL-31-IgG. The cells were cultured and
phase-contrast images of microvessel tubes were captured after 200
min at 100.times. magnification using the Leica CTR 6000 (Leica
Microsystems). The images were analyzed using ImageJ software and
quantified by counting the number of HUVEC junctions (bifurcations)
per field. FIG. 9A shows representative images of tube forming of
HUVECs in the presence of 100 ng/ml recombinant human IL-31
(rhIL31) or 10 .mu.g/ml IL-31-IgG are provided 200 min time-point.
The number of bifurcations per field were quantified and presented.
*, p<0.05; ***, p<0.001 as shown in FIG. 9B.
DETAILED DESCRIPTION OF THE INVENTION
[0047] In the following detailed description, numerous specific
details are set forth in order to provide a thorough understanding
of the invention. However, it will be understood by those skilled
in the art that the present invention may be practiced without
these specific details. In other instances, well-known methods,
procedures, and components have not been described in detail so as
not to obscure the present invention.
[0048] As shown in the Examples section, IL-31 was found to be
highly effective in inhibiting tumor cells proliferation, tumor
size and metastasis in cells and tumors. In an embodiment of the
invention, the benefit of IL-31 in tumors may be dependent on the
presence of IL-31 or IL-31R expression in the tumor. In some
embodiments of the invention, as exemplified in Examples 2 and 4,
IL-31 may have an indirect effect on any tumor cell, for example,
via an effect on supporting cells in the tumor microenvironment
i.e., endothelial cells and macrophages In some embodiments of the
invention, there is provided a method for treating cancer
comprising the step of administering IL-31, a fused protein
comprising IL-31, or a complex comprising either IL-31 or a fused
protein that comprises IL-31 to a subject in need, thereby treating
cancer.
[0049] In some embodiments of the invention, there is provided a
method for treating cancer comprising the step of contacting
cancerous cells of the subject with a therapeutically effective
amount of IL-31, a fused protein comprising IL-31, or a complex
comprising either IL-31 or a fused protein that comprises IL-31,
thereby treating the cancer.
[0050] In some embodiments of the invention, there is provided a
method for treating cancer comprising the step of contacting
cancerous cells of the subject with a therapeutically effective
amount of an agent capable of up-regulating IL-31 or an agent that
is an agonist to IL-31 receptors, thereby treating the cancer.
[0051] According to some embodiments of the invention, the
contacting is effected in-vivo.
[0052] According to some embodiments of the invention, the
contacting is effected ex-vivo.
[0053] In some embodiments of the invention, the cancer is an oral
cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory
cancer, a urogenital cancer, a gastrointestinal cancer, a central
or peripheral nervous system tissue cancer, an endocrine or
neuroendocrine cancer or a hematopoietic cancer.
[0054] According to some embodiments of the invention, the cancer
is a glioma, a sarcoma, a carcinoma, a lymphoma, a melanoma, a
fibroma, or a meningioma.
[0055] According to some embodiments of the invention, the cancer
is brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal
cancer, biliary cancer, prostatic cancer, pheochromocytoma,
pancreatic islet cell cancer, Li-Fraumeni tumors, thyroid cancer,
parathyroid cancer, pituitary tumors, adrenal gland tumors,
osteogenic sarcoma tumors, multiple neuroendrcine type I and type
II tumors, breast cancer, lung cancer, head & neck cancer,
prostate cancer, esophageal cancer, tracheal cancer, skin cancer
brain cancer, liver cancer, bladder cancer, stomach cancer,
pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer,
testicular cancer, colon cancer, rectal cancer or skin cancer.
[0056] In some embodiments of the invention, the cancer is a breast
cancer, a pancreatic cancer or a lung cancer.
[0057] In some embodiments of the invention, there is provided a
method of preventing or reducing metastasis comprising the step of
administering a therapeutically effective amount of IL-31, a fused
protein comprising IL-31, an agent the up-regulates IL-31 or an
agent that is an agonist to IL-31 receptor or a complex comprising
either IL-31 or a fused protein that comprises IL-31, to a subject
in need thereby preventing metastasis.
[0058] As used herein the term "treating cancer" refers to
preventing, curing, reversing, attenuating, alleviating, minimizing
or suppressing the cancer, as well as resulting in one or more of
the following parameters: reduction in tumor size or burden,
blocking of tumor growth, shifting the phenotype of the macrophage
from M2 to M1, reduction in tumor-associated pain, long-term
non-progression, induction of remission, reduction of metastasis,
or increased patient survival.
[0059] As used herein the term "cancer" refers to the presence of
cells possessing characteristics typical of cancer-causing cells,
for example, uncontrolled proliferation, loss of specialized
functions, immortality, significant metastatic potential,
significant increase in anti-apoptotic activity, rapid growth and
proliferation rate, and certain characteristic morphology and
cellular markers. Typically, the cancer cells are in the form of a
tumor; existing locally within an animal, or circulating in the
blood stream as independent cells, for example, leukemic cells.
[0060] The method may further comprise administering a second
anti-cancer therapy or a third anti-cancer therapy to the treated
subject. The second and third anti-cancer therapies may be one or
two or more of chemotherapy, radiotherapy, hormonal therapy,
cytokine therapy, immunotherapy, targeted therapy, e.g.,
bortezomib, sunitinib, Herceptin, sorafenib and/or surgery. The
second and third anti-cancer therapy may be administered to the
subject prior to or after the IL-31 treatment or concurrent with
the IL-31 treatment. As used herein, the IL-31 treatment includes
treatments using IL-31, a fused protein comprising the same, IL-31
receptor agonist or an agent which up-regulates IL-31 or a complex
comprising IL-31 or a fused protein thereof.
[0061] In some embodiments, the method of treatment may further
comprise assessing the efficacy of the treatment by performing a
PET scan on said subject or measuring the level of the relevant
bio-markers.
[0062] In some embodiments of the invention, there is provided a
method for treating angiogenesis related disorders or diseases
comprising the step of administering a therapeutically effective
amount of IL-31, a fused protein comprising IL-31, or a complex
comprising either IL-31 or a fused protein that comprises IL-31 or
an agent capable of upregulating IL-31 to a subject in need thereby
treating an angiogenesis related disorder or disease.
[0063] As used herein the term "treating angiogenesis relate
disorders" refers to preventing, curing, reversing, attenuating,
alleviating, minimizing or suppressing the angiogenesis related
disorders, as well as resulting in a decrease of abnormal or
pathological angiogenesis or increased efficacy of functional
angiogenesis by inhibiting permeabilization and thus increased
patient survival or reduce symptoms. The method may further
comprise administering an additional therapy prior to or after the
IL-31 treatment or concurrent with the IL-31 treatment to the
treated subject, such as, hormonal therapy, another
anti-angiogenesis therapy, immunotherapy or a targeted therapy to
the abnormal angiogenesis related disease.
[0064] According to some embodiments of the invention, the disease
associated with angiogenesis is selected from the group consisting
of cancer, arthritis, rheumatoid arthritis, atherosclerotic
plaques, corneal graft neovascularization, hypertrophic or keloid
scars, proliferative retinopathy, diabetic retinopathy, macular
degeneration or age related macular degeneration (AMD),
granulation, neovascular glaucoma and uveitis. In some embodiments,
the angiogenesis related disorder is "fibrosis-related diseases,
e.g., liver fibrosis and lung fibrosis. In some embodiments, the
angiogenesis related disorder is asthma. In some embodiments, the
angiogenesis related disorder is Idiopathic Pulmonary Fibrosis
(IPF) and Myelofibrosis, Primary Sclerosing Cholangitis.
[0065] In some embodiments of the invention, angiogenesis-related
diseases include, but are not limited to, inflammatory, autoimmune,
and infectious diseases; angiogenesis-dependent cancer, including,
for example, solid tumors, blood born tumors such as leukemias, and
tumor metastases; benign tumors, for example hemangiomas, acoustic
neuromas, neurofibromas, trachomas, and pyogenic granulomas;
psoriasis; eczema; ocular angiogenic diseases, for example,
diabetic retinopathy, retinopathy of prematurity, corneal graft
rejection, retrolental fibroplasia, rubeosis; Osler-Webber
Syndrome; myocardial angiogenesis; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; and wound
granulation.
[0066] In addition, compositions comprising the active ingredient
as defined herein can be used to treat diseases such as, but not
limited to, intestinal adhesions, atherosclerosis, scleroderma,
warts, and hypertrophic scars (i.e., keloids). Compositions of this
invention may also be useful in the treatment of diseases that have
angiogenesis as a pathologic consequence, such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helobacter pylori),
tuberculosis, and leprosy. In some embodiments, the compositions
which include the active ingredient of the invention may be used to
treat inflammation or inflammation related disorders.
[0067] For example, human IL-31 (SEQ ID NO: 1 below) (Gene ID:
386653) is encoded by the following nucleic acid sequence:
TABLE-US-00001 (SEQ ID NO: 2)
ATGGCCTCTCACTCAGGCCCCTCGACGTCTGTGCTCTTTCTGTTCTGCTG
CCTGGGAGGCTGGCTGGCCTCCCACACGTTGCCCGTCCGTTTACTACGAC
CAAGTGATGATGTACAGAAAATAGTCGAGGAATTACAGTCCCTCTCGAAG
ATGCTTTTGAAAGATGTGGAGGAAGAGAAGGGCGTGCTCGTGTCCCAGAA
TTACACGCTGCCGTGTCTCAGCCCTGACGCCCAGCCGCCAAACAACATCC
ACAGCCCAGCCATCCGGGCATATCTCAAGACAATCAGACAGCTAGACAAC
AAATCTGTTATTGATGAGATCATAGAGCACCTCGACAAACTCATATTTCA
AGATGCACCAGAAACAAACATTTCTGTGCCAACAGACACCCATGAATGTA
AACGCTTCATCCTGACTATTTCTCAACAGTTTTCAGAGTGCATGGACCTC
GCACTAAAATCATTGACCTCTGGAGCCCAACAGGCCACCACTTAA.
Human IL-31 Amino Acid Sequence:
TABLE-US-00002 [0068] (1-23: Signal peptide; 24-164: IL-31) (SEQ ID
NO: 1) MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSK
MLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDN
KSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDL
ALKSLTSGAQQATT
[0069] For example, mouse (Mus musculus) IL-31 (SEQ ID NO: 6 below)
(Gene ID: 76399) is encoded by the following nucleic acid
sequence:
TABLE-US-00003 (SEQ ID NO: 3)
ATGATCTTCCACACAGGAACAACGAAGCCTACCCTGGTGCTGCTTTGCTG
TATAGGAACCTGGCTGGCCACCTGCAGCTTGTCCTTCGGTGCCCCAATAT
CGAAGGAAGACTTAAGAACTACAATTGACCTCTTGAAACAAGAGTCTCAG
GATCTTTATAACAACTATAGCATAAAGCAGGCATCTGGGATGTCAGCAGA
CGAATCAATACAGCTGCCGTGTTTCAGCCTGGACCGGGAAGCATTAACCA
ACATCTCGGTCATCATAGCACATCTGGAGAAAGTCAAAGTGTTGAGCGAG
AACACAGTAGATACTTCTTGGGTGATAAGATGGCTAACAAACATCAGCTG
TTTCAACCCACTGAATTTAAACATTTCTGTGCCTGGAAATACTGATGAAT
CCTATGATTGTAAAGTGTTCGTGCTTACGGTTTTAAAGCAGTTCTCAAAC
TGCATGGCAGAACTGCAGGCTAAGGACAATACTACATGCTGA.
Mouse IL-31 Amino Acid Sequence:
TABLE-US-00004 [0070] (1-23: Signal peptide; 24-163: IL-31) (SEQ ID
NO: 6) MIFHTGTTKPTLVLLCCIGTWLATCSLSFGAPISKEDLRTTIDLLKQESQ
DLYNNYSIKQASGMSADESIQLPCFSLDREALTNISVIIAHLEKVKVLSE
NTVDTSWVIRWLTNISCFNPLNLNISVPGNTDESYDCKVFVLTVLKQFSN
CMAELQAKDNTTC
[0071] Specifically, IL-31 SEQ ID. No. 1 (Gene ID. No. 386653 (for
human), Gene ID. No 76399 (for Mus Musculus), Gene ID. No 744097
(for Pan Troglodytes (chimpanzee)), Gene ID. No. 102179123 (for
Capra Hircus (goat)), which form a part of the invention also
refers to homologs (e.g., polypeptides), which are at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 87%, at least about 89%, at least about
91%, at least about 93%, at least about 95%, at least about 97% or
more, homologous to the IL-31 sequence as set forth in SEQ ID No. 1
listed herein, as determined using any appropriate means, including
BlastP software of the National Center of Biotechnology Information
(NCBI) using default parameters). The homolog may also refer to a
deletion, insertion, or substitution variant, including an amino
acid substitution, thereof and biologically active polypeptide
fragments thereof.
[0072] As used herein the term "about" refers to .+-.10%.
[0073] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically or
pharmaceutically suitable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration of a
compound to an organism.
[0074] Herein the term "active ingredient" refers to IL-31 or a
fused protein comprising IL-31 or to a complex comprising the IL-31
or the fused protein comprising IL-31, or to the agent capable of
up-regulating IL-31, or an IL-31 receptor agonist any one of which
is accountable for biological effect as described herein. Further
included are constructs which include nucleic acid encoding the
same.
[0075] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier", which may be
interchangeably used, refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0076] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0077] In some embodiments, the invention further envisages
inclusion of the IL-31 sequence or a fused protein thereof in a
complex where it is attached to proteinaceous (e.g., heterologous
amino acid sequence) or non-proteinaceous moieties (e.g., PEG),
each of which being capable of prolonging the half-life of the
composition while in circulation.
[0078] Such a molecule is highly stable (resistant to in-vivo
proteaolytic activity, probably due to steric hindrance conferred
by the non-proteinaceous moiety) and may be produced using common
solid phase synthesis. Further recombinant techniques may still be
used, whereby the recombinant peptide product is subjected to
in-vitro modification (e.g., PEGylation as further described herein
below).
[0079] The phrase "non-proteinaceous moiety" as used herein refers
to a molecule not including peptide bonded amino acids that is
attached to the above-described IL-31 amino acid sequence.
According to some embodiments the non-proteinaceous moiety may be a
polymer or a co-polymer (synthetic or natural). Non-limiting
examples of the non-proteinaceous moiety of the present invention
include polyethylene glycol (PEG) or derivative thereof, Polyvinyl
pyrrolidone (PVP), albumin, divinyl ether and maleic anhydride
copolymer (DIVEMA); polysialic acid (PSA) and/or poly(styrene
comaleic anhydride) (SMA). Additionally, complexes which can
protect IL-31 from the environment and thus keep its stability may
be used, including, for example, liposomes or micelles containing
IL-31, IL-31 receptor agonist, an agent that up-regulates IL-31 or
a fused protein comprising thereof are also included in the
invention.
[0080] By "an agent that is an agonist to IL-31 receptors" or IL-31
receptor agonist" it is meant any agent that binds to an IL-31
receptor and produce a biological response as defined herein. Such
an agent may be a protein, a small molecule, an antibody and the
like.
[0081] According to some embodiments of the invention, the IL-31 or
the fused protein comprising IL-31 of the invention is attached to
a non-proteinaceous moiety, which may act as a sustained-release
enhancing agent. Exemplary sustained-release enhancing agents
include, but are not limited to hyaluronic acid (HA), alginic acid
(AA), polyhydroxyethyl methacrylate (Poly-HEMA), glyme and
polyisopropylacrylamide.
[0082] Attaching the amino acid sequence component of the IL-31 or
the fused protein comprising thereof of the invention to other
non-amino acid agents may be by covalent linking or by non-covalent
complexion, for example, by complexion to a hydrophobic polymer,
which can be degraded or cleaved producing a compound capable of
sustained release; by entrapping the amino acid part of the IL-31
or the fused protein comprising thereof in liposomes or micelles to
produce a complex comprising the IL-31 or the fused protein
comprising the same. The association may be by the entrapment of
the amino acid sequence within the other component (liposome,
micelle) or the impregnation of the amino acid sequence within a
polymer to produce the final peptide of the invention.
[0083] In some embodiments, the PEG derivative is
N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids,
succinimidyl ester of carboxymethylated PEG (SCM-PEG),
benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG,
PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC),
PEG aldehydes, PEG-orthopyridyl-disulfide,
carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide.
PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC) or
PEG-orthopyridyl disulfide may be also used.
[0084] The non-proteinaceous moiety may be attached to the IL-31
amino acid sequence in any chosen position, provided that the
therapeutic activity of IL-31 is retained.
[0085] In some embodiments, the conjugated IL-31 molecules are
separated, purified and qualified using e.g., high-performance
liquid chromatography (HPLC).
[0086] Molecules of this aspect of the present invention may be
biochemically synthesized such as by using standard solid phase
techniques. These methods include exclusive solid phase synthesis,
partial solid phase synthesis methods, fragment condensation and
classical solution synthesis.
[0087] Solid phase peptide synthesis procedures are well known in
the art and further described by John Morrow Stewart and Janis
Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0088] In instances where large amounts of the peptides of the
present invention are desired, they may be produced using
recombinant techniques such as described by Bitter et al. (1987)
Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in
Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514;
Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984)
EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843;
Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach &
Weissbach, 1988&, Methods for Plant Molecular Biology, Academic
Press, NY, Section VIII, pp 421-463.
[0089] In some embodiments of the invention, there is provided a
fused protein that comprises IL-31 as defined herein together with
one or more molecule which extend the half life of IL-31 in the
plasma. In some embodiments, the fused protein further comprises a
linker. In some embodiments of the invention, there is provided a
fused protein that comprises IL-31 and a protein that stabilizes
IL-31 or protect it in the blood stream or at the tissue. In some
embodiments the fused protein comprises IL-31 attached to a
heterologous amino acid sequence. In some embodiments, the
heterologous amino acid sequence comprises an immunoglobulin amino
acid sequence.
[0090] In some embodiments of the invention, there is provided a
fused protein that comprises IL-31 and IgG. The IgG may any
subclasses or isotypes thereof, e.g., IgG1, IgG2, IgG3, IgG4. For
example:
Mus musculus Immunoglobulin Gamma Heavy Chain (Partial Cds of
DQ381548)
TABLE-US-00005 (SEQ ID NO: 4)
GTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGT
ATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTA
CTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGAT
CCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGC
TCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCA
GTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAA
TGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTC
CAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTC
CCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACA
GACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCC
AGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTT
ACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGA
AATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATAC
TGAGAAGAGCCTCTCCCACTCTCCTGGTAAA.
Homo sapiens mRNA for IgG H Chain, (Partial Cds of AB776838)
TABLE-US-00006 (SEQ ID NO: 5)
GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG
ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTG
ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.
[0091] In some embodiments, the term "antibody" refers to the
structure that constitutes the natural biological form of an
antibody. In most mammals, including humans, and mice, this form is
a tetramer and consists of two identical pairs of two
immunoglobulin chains, each pair having one light and one heavy
chain, each light chain comprising immunoglobulin domains V.sub.L
and C.sub.L, and each heavy chain comprising immunoglobulin domains
V.sub.H, C.gamma.1, C.gamma.2, and C.gamma.3. In each pair, the
light and heavy chain variable regions (V.sub.L and V.sub.H) are
together responsible for binding to an antigen, and the constant
regions (C.sub.L, C.gamma.1, C.gamma.2, and C.gamma.3, particularly
C.gamma.2, and C.gamma.3) are responsible for antibody effector
functions. In some mammals, for example in camels and llamas,
full-length antibodies may consist of only two heavy chains, each
heavy chain comprising immunoglobulin domains V.sub.H, C.gamma.2,
and C.gamma.3. By "immunoglobulin (Ig)" herein is meant a protein
consisting of one or more polypeptides substantially encoded by
immunoglobulin genes. Immunoglobulins include but are not limited
to antibodies Immunoglobulins may have a number of structural
forms, including but not limited to full-length antibodies,
antibody fragments, and individual immunoglobulin domains including
but not limited to V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3,
V.sub.L, C.sub.L, Fab and Fc fragments.
[0092] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five-major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called alpha,
delta, epsilon, gamma, and mu, respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0093] In some embodiments, a construct which includes a nucleic
acid sequence for IL-31 as defined herein, for example, SEQ ID. No.
2, and a nucleic acid sequence for IgG for example, SEQ ID. No. 5
may be used in the construct. In some embodiments of the invention,
the IL-31 and the IgG are directly fused to each other.
[0094] Serum albumin can also be engaged in half-life extension
through modules with the capacity to non-covalently interact with
albumin. In these approaches, an albumin-binding moiety is either
conjugated or genetically fused to the therapeutic protein Proteins
with albumin-binding activity are known from certain bacteria. For
example, streptococcal protein G contains several small
albumin-binding domains (ABD) composed of roughly 50 amino acid
residues (6 kDa). Fusion of an ABD to a protein results in a
strongly extended half-life (see Roland E Kontermann, trategies for
extended serum half-life of protein therapeutics, Current Opinion
in Biotechnology 2011, 22:868-876.
[0095] In some embodiments of the invention, the IL-31 and the IgG
and/or any other protein that may be used for extending the
half-life of IL-31 in the serum are linked by a linker. In Some
embodiments of the invention, the linker is a sequence of between
2-20 amino acids.
[0096] In Some embodiments of the invention, the linker is a
sequence of between 4-12 amino acids which form a cleavage site for
enzymes such as MMP9/2, trypsin, PSA, cathepsins, kallikreins,
serine proteases, caspases and others. Additional possible cleavage
sites are presented in CHOI et al., "Protease-Activated Drug
Development", Theranostics, Vol. 2(2), pp. 156-178 (found in
http://www.thno.org/v02p0156.pdf). In some embodiments, the linker
is between 6-8 amino acids and in some embodiments includes a
cleavage site for enzymes such as MMP9/2, trypsin, PSA, cathepsins,
kallikreins, serine proteases, caspases and/or others.
[0097] In some embodiments, the linker that comprise a cleavage
site of MMP-9/2, cathepsin, trypsin, kallikreins, serine proteases,
caspases or any other cleaving enzyme that can be added between
IL-31 and IgG. For example, a sequence of the following amino acids
between IL-31 and IgG may be provided:
-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln- (SEQ ID: No. 7, for MMP-9/2
cleaving site), -Lys-Lys-Phe-D-Ala-.epsilon.-maleimidocaproic acid
(SEQ ID. No: 8, for cathepsin B cleaving site);
-Lys-Gly-Ala-Ser-D-Arg-Phe-Thr-Gly--(SEQ ID: No. 9, for trypsin
cleaving site); or .epsilon.-maleimidocaproic
acid-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly (SEQ ID No: 10, for PSA
cleaving site).
[0098] Furthermore, the present invention encompasses nucleic acids
encoding the fusion proteins described herein. In addition, vectors
comprising these nucleic acids and cells transformed with theses
vectors are encompassed by the present invention.
[0099] Briefly, the fused protein is prepared as follows: an
expression construct (i.e., expression vector), which includes an
isolated polynucleotide (i.e., isolated from a naturally occurring
source thereof, e.g., SEQ ID NO: 2 or SEQ ID NO: 3 Gene ID 386653
(for human) and 76399 (for mouse) that comprises a nucleic acid
sequence encoding the IL-31 amino acid sequence fused (optionally
including a linker) in frame to a nucleic acid sequence encoding
the IgG amino acid sequence e.g., AB776838 (for human, NCBI
database) or DQ38154 (for mouse, NCBI database) or SEQ ID. No: 5 or
SEQ ID. No. 4, respectively, positioned under the transcriptional
control of a regulatory element, such as a promoter, is introduced
into host cells.
[0100] For example, a nucleic acid sequence encoding an IL-31 amino
acid sequence of the invention (e.g., SEQ ID NO:1 or SEQ ID NO:6,
Gene ID. 386653 (for human) or 76399 (for mouse) is ligated
in-frame to an immunoglobulin cDNA sequence (e.g., AB776838 (for
human) and DQ38154 (for mouse).
[0101] In some embodiments of the invention, when a cleaving site
for enzymes is required, a nucleic acid sequence encoding e.g. one
or more of the amino acid sequences SEQ ID. Nos. 7-10, is added to
the construct.
[0102] It will be appreciated that, ligation of genomic
immunoglobulin fragments can also be used. In this case, fusion
requires the presence of immunoglobulin regulatory sequences for
expression. cDNAs encoding IgG heavy-chain constant regions can be
isolated based on published sequences from cDNA libraries, derived
from spleen or peripheral blood lymphocytes, by hybridization or by
polymerase chain reaction (PCR) techniques. The nucleic acid
sequences encoding the IL-31 amino acid sequence and immunoglobulin
can be ligated in tandem into an expression construct (vector) that
directs efficient expression in the selected host cells, further
described hereinbelow. For expression in mammalian cells,
pRK5-based vectors [Schall et al., Cell, 61:361-370 (1990)]; and
CDM8-based vectors [Seed, Nature, 329:840 (1989)] can be used. The
exact junction can be created by removing the extra sequences
between the designed junction codons using oligonucleotide-directed
deletional mutagenesis [Zoller et al, Nucleic Acids Res., 10:6487
(1982); Capon et al., Nature, 337:525-531 (1989)]. Synthetic
oligonucleotides can be used, in which each half is complementary
to the sequence on either side of the desired junction; ideally,
these are 11 to 48-mers. Alternatively, PCR techniques can be used
to join the two parts of the molecule in-frame with an appropriate
vector.
[0103] Methods of introducing the expression construct into a host
cell are well known in the art and include electroporation,
lipofection and chemical transformation (e.g., calcium phosphate).
See also Example 5 of the Examples section which follows, as well
as in the Experimental procedures section therein.
[0104] The "transformed" cells are cultured under suitable
conditions, which allow the expression of the chimeric molecule
encoded by the nucleic acid sequence.
[0105] Following a predetermined time period, the expressed
chimeric molecule is recovered from the cell or cell culture, and
purification is effected according to the end use of the
recombinant polypeptide.
[0106] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements including
constitutive and inducible promoters, transcription enhancer
elements, transcription terminators, and the like, can be used in
the expression vector [see, e.g., Bitter et al., (1987) Methods in
Enzymol. 153:516-544].
[0107] Other than containing the necessary elements for the
transcription and translation of the inserted coding sequence
(encoding the chimera), the expression construct of the present
invention can also include sequences engineered to optimize
stability, production, purification, yield or effectiveness of the
expressed fusion protein.
[0108] A variety of prokaryotic or eukaryotic cells can be used as
host-expression systems to express the fusion protein coding
sequence. These include, but are not limited to, microorganisms,
such as bacteria transformed with a recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vector containing the chimera
coding sequence; yeast transformed with recombinant yeast
expression vectors containing the chimera coding sequence; plant
cell systems infected with recombinant virus expression vectors
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or transformed with recombinant plasmid expression vectors, such as
Ti plasmid, containing the chimera coding sequence. Mammalian
expression systems may also be used to express the chimera of the
invention.
[0109] The choice of host cell line for the expression of the
molecules depends mainly on the expression vector. Eukaroyotic
expression systems are preferred (e.g., mammalian and insects)
since they allow post translational modifications (e.g.,
glycosylation). Another consideration is the amount of protein that
is required. Milligram quantities often can be produced by
transient transfections. For example, the adenovirus
EIA-transformed 293 human embryonic kidney cell line can be
transfected transiently with pRK5-based vectors by a modification
of the calcium phosphate method to allow efficient expression.
CDM8-based vectors can be used to transfect COS cells by the
DEAE-dextran method (Aruffo et al., Cell, 61:1303-1313 (1990);
Zettmeissl et al., DNA Cell Biol. US, 9:347-353 (1990)]. If larger
amounts of protein are desired, the molecules can be expressed
after stable transfection of a host cell line (see Example 1 of the
Examples section). It will be appreciated that the presence of a
hydrophobic leader sequence at the N-terminus of the molecule will
ensure processing and secretion of the molecule by the transfected
cells.
[0110] It will be appreciated that the use of bacterial or yeast
host systems may be preferable to reduce cost of production.
However since bacterial host systems are devoid of protein
glycosylation mechanisms, a post production glycosylation may be
needed.
[0111] According to some embodiments, transformed cells are
cultured under effective conditions, which allow for the expression
of high amounts of recombinant polypeptide. Effective culture
conditions include, but are not limited to, effective media,
bioreactor, temperature, pH and oxygen conditions that permit
protein production. An effective medium refers to any medium in
which a cell is cultured to produce the recombinant chimera
molecule of the present invention. Such a medium typically includes
an aqueous solution having assimilable carbon, nitrogen and
phosphate sources, and appropriate salts, minerals, metals and
other nutrients, such as vitamins. Cells of the present invention
can be cultured in conventional fermentation bioreactors, shake
flasks, test tubes, microtiter dishes, and petri plates. Culturing
can be carried out at a temperature, pH and oxygen content
appropriate for a recombinant cell. Such culturing conditions are
within the expertise of one of ordinary skill in the art.
[0112] Depending on the vector and host system used for production,
resultant proteins of the present invention may either remain
within the recombinant cell, secreted into the fermentation medium,
secreted into a space between two cellular membranes, such as the
periplasmic space in E. coli; or retained on the outer surface of a
cell or viral membrane.
[0113] Following a predetermined time in culture, recovery of the
recombinant protein is affected. The phrase "recovering the
recombinant protein" refers to collecting the whole fermentation
medium containing the protein and need not imply additional steps
of separation or purification. Proteins of the present invention
can be purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity chromatography,
ion exchange chromatography, filtration, electrophoresis,
hydrophobic interaction chromatography, gel filtration
chromatography, reverse phase chromatography, concanavalin A
chromatography, chromatofocusing and differential
solubilization.
[0114] Molecules of the present invention are preferably retrieved
in "substantially pure" form. As used herein, "substantially pure"
refers to a purity that allows for the effective use of the protein
in the applications, described herein below.
[0115] Recombinant molecules of the present invention can be
conveniently purified by affinity chromatography. The suitability
of protein A as an affinity ligand depends on the species and
isotype of the immunoglobulin Fc domain that is used in the
chimera. Protein A can be used to purify chimeric molecules that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
[Lindmark et al., J. Immunol. Meth., 62:1-13 (1983)]. Protein G is
preferably used for all mouse isotypes and for human .gamma.3 [Guss
et al., EMBO J., 5:1567-1575 (1986)]. The solid support to which
the affinity ligand is attached is most often agarose, but other
solid supports are also available. Mechanically stable solid
supports such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. The conditions
for binding the chimeric molecules to the protein A or G affinity
column are dictated entirely by the characteristics of the Fc
domain; that is, its species and isotype. Generally, when the
proper ligand is chosen, efficient binding occurs directly from
unconditioned culture fluid. One distinguishing feature of chimeric
molecules of this aspect of the present invention is that, for
human .gamma.1 molecules, the binding capacity for protein A is
somewhat diminished relative to an antibody of the same Fc type.
Bound chimeric molecules of this aspect of the present invention
can be efficiently eluted either at acidic pH (at or above 3.0), or
in a neutral pH buffer containing a mildly chaotropic salt. This
affinity chromatography step can result in a chimeric molecule
preparation that is >95% pure. Medical grade purity is essential
for therapeutic applications.
[0116] Other methods known in the art can be used in place of, or
in addition to, affinity chromatography on protein A or G to purify
chimeric molecules which include an immunoglobulin portion. Such
chimeric molecules behave similarly to antibodies in thiophilic gel
chromatography [Hutchens et al., Anal. Biochem., 159:217-226
(1986)] and immobilized metal chelate chromatography [Al-Mashikhi
et al., J. Dairy Sci., 71:1756-1763 (1988)]. In contrast to
antibodies, however, their behavior on ion exchange columns is
dictated not only by their isoelectric points, but also by a charge
dipole that may exist in the molecules due to their chimeric
nature.
[0117] The above-described molecules are preferably non-immunogenic
for maximizing therapeutic efficacy.
[0118] As used herein the term "non-immunogenic" refers to a
substance that is substantially incapable of producing an immune
response in a subject administered therewith. For example,
non-immunogenic in a human means that upon contacting the chimeric
molecule of this aspect of the present invention with the
appropriate tissue of a human, no state of sensitivity or
resistance to the chimeric molecule is demonstrable upon the second
administration of the chimeric molecule after an appropriate latent
period (e.g., 8 to 14 days).
[0119] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0120] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
inrtaperitoneal, intranasal, or intraocular injections.
[0121] Alternately, one may administer the pharmaceutical
composition in a local, rather than systemic, manner, for example,
via injection of the pharmaceutical composition directly into a
specific tissue region of a patient.
[0122] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0123] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0124] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0125] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0126] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0127] Pharmaceutical compositions that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules may contain the active ingredients in
admixture with fillers such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0128] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0129] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0130] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0131] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0132] According to some embodiments of the invention, the active
ingredient may be in powder form for constitution with a suitable
vehicle, e.g., sterile, pyrogen-free water based solution, before
use.
[0133] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0134] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients (IL-31) effective to
prevent, alleviate or ameliorate symptoms of a disorder
(angiogenesis related disease or cancer) or prolong the survival of
the subject being treated.
[0135] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, and depends on
the severity of the disease, its type, the mode of administration
and the like.
[0136] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0137] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in humans. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0138] Dosage amount and interval may be adjusted individually to
ensure levels of the active ingredient are sufficient to induce or
suppress the biological effect (minimal effective concentration,
MEC). The MEC will vary for each preparation, but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
Detection assays can be used to determine plasma
concentrations.
[0139] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0140] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc. Compositions of the present invention
may, if desired, be presented in a pack or dispenser device, such
as an FDA approved kit, which may contain one or more unit dosage
forms containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accommodated by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
preparation of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as is further detailed above.
EXAMPLES
Experimental Procedures Used in the Examples
Cell Culture
[0141] MC38 murine colon carcinoma, and 4T1 murine breast carcinoma
cell lines (ATCC, Manassas, Va., USA) and were used within 6 months
of resuscitation. The cell lines were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 1%
L-glutamine, 1% sodium pyruvate, and 1% streptomycin, penicillin
and neomycin in solution (10 mg/ml, Biological Industries,
Israel).
Cell Viability Alamar Blue.TM. Assay
[0142] Cell viability was evaluated quantitatively with the
metabolic indicator dye AlamarBlue.TM. (Serotec Ltd., Oxford, UK),
which determines the metabolic activity of cells and is used for
cell viability and proliferation as previously described in
Voloshin T, Gingis-Velitski S, Bril R, Benayoun L, Munster M,
Milsom C, Man S, Kerbel R S, Shaked Y (2011) G-CSF supplementation
with chemotherapy can promote revascularization and subsequent
tumor regrowth: prevention by a CXCR4 antagonist (Blood 118
(12):3426-3435). Cells were harvested from sub-confluent cultures
and re-plated (500-1,000 cells/well in a 96-well plate) in their
designated medium and 10% AlamarBlue (AB) solution. In some
experiments mouse or human recombinant IL-31 (Peprotek, Israel) was
added in escalating concentrations (usually 0.5 ng-20 ng/ml).
Results were corrected to background values of negative controls.
Results are presented as a percent reduction of AB, calculated by
the appropriate equation. All experiments were performed in
triplicate, and data were presented as means.+-.standard error.
Western Blot
[0143] Fifty .mu.g of proteins were analyzed by 10% SDS/PAGE gel,
transferred to a nitrocellulose membrane, and subsequently blotted
with mouse IL-31 antibody (5 .mu.g/ml, Abcam) or human IL-31
antibody (5 .mu.g/ml, Abcam), and normalized with actin mouse
monoclonal antibody (1:5,000, MP Biomedicals).
Downregulation of IL-31 by shRNA
[0144] Four sequence shRNA clones specific to murine IL-31 or shRNA
control (empty scrambled vector) were constructed by Applied
Biological Materials (Canada). The cells were transfected at a
confluency of 60%. Transfection was achieved using FuGENEV R 6
(Roche, Penzberg, Germany) according to the manufacturer's
instructions. 48 hours post-transfection, cells were incubated in
growth medium containing puromycin (1 .mu.l/ml) to select for
stable transfectants. After two weeks of selection, the IL-31
shRNA-mediated gene silencing was assessed for each clone using
western blot analysis.
Flow Cytometry
[0145] Tumor cells, macrophages, or cells obtained from tumors
followed by single cell suspension procedure as previously
described in Adini A, Fainaru O, Udagawa T, Connor K M, Folkman J,
D'Amato R J (2009) Matrigel cytometry: a novel method for
quantifying angiogenesis in vivo. J Immunol. Methods 342
(1-2):78-81, were immunostained with the following antibody
mixtures: For the analysis of endothelial cells:
CD45-/CD31+/VEGFR2+; for MDSCs: Gr-1+, CD11b+; for M1 macrophages
(F4/80+CD11c+CD206-) and for M2 macrophages (F4/80+CD11c-CD206+).
All antibodies were purchased from BD Biosciences or BioLegend (San
Diego, Calif.). The experiments were performed on Cyan-ADP flow
cytometer (Beckman Coulter, Switzerland) and analyzed with Summit
Version 4.3 (Beckman Coulter).
Tissue Processing and Immunostaining
[0146] Tumors (were embedded in OCT and were subsequently
sectioned. 10 .mu.m cryosections were used for the analysis of
microvessel density (MVD) and macrophage colonization in the
tissue. For endothelial cells, anti-CD31 antibody was used as a
specific endothelial marker (1:200, BD Biosciences) along with a
Cy3-conjugated secondary antibody (1:500, Jackson immunoresearch
laboratories). For macrophages, anti-F4/80 conjugated to FITC
antibody was used as a specific macrophage marker (1:200, BD
Biosciences). The number of vessel structures or cells per field
were counted and plotted. (At least 5 fields per tumor, n>20
fields per group).
Tumor Models
[0147] MC38 murine-colorectal carcinoma cells (2.times.10.sup.6)
were subcutaneously injected into the flank of 5- to 6-week-old
female BALB/c mice (Harlan, Israel). 4T1 murine-breast carcinoma
cells (5.times.10.sup.5) were orthotopically injected into the
mammary fat pad of 6-week-old female BALB/c mice. Tumor size was
assessed regularly with Vernier calipers using the formula
width.sup.2.times.length.times.0.5. All animal studies were
performed in accordance with the Animal Care and Use Committee of
the Technion-Israel Institute of Technology.
Osmotic Pumps
[0148] Osmotic minipumps were used in vivo to allow the continuous
administration of recombinant IL-31 to mice for a period of two
weeks, as per the manufacturer's instructions. Briefly, when tumors
reached 150-200 mm.sup.3, treatment was initiated by subcutaneous
implantation of osmotic minipump (#1002, Alzet, Cupertino, Calif.)
loaded with recombinant mouse IL-31 (in a total concentration of
0.7 .mu.g per day) or PBS as a vehicle control. The procedure was
performed under sterile conditions. The mice were sacrificed 14
days after pump implantation and further assessed as described in
the text.
IL-31-IgG Construct
[0149] The corresponding DNA sequence of mature IL-31 protein with
its signal peptide for secretion was synthesized based on gBlocks
Gene Fragment technology from IDT, and inserted into NSPI
expression vector. Mouse IgG1 heavy chain (hinge-CH2-CH3) was
cloned downstream to IL-31 and upstream to myc-His6. Briefly, total
RNA was isolated from mouse spleen using RNeasy Mini Kit (Qiagen).
Single-stranded cDNA was synthesized using M-MLV reverse
transcriptase (Promega) according to manufacturer's instructions.
Mouse IgG1 heavy chain (hinge-CH2-CH3) was amplified using PCR. The
primers used were: sense (5-TACCGCTCGAGGTGCCCAGGGATTGTGGTTG-3) (SEQ
ID NO: 11) and antisense (5'-CGTTCGAATTTACCAGGAGAGTGGG-3) (SEQ ID
NO: 12). The PCR fragment provides a size of .about.700 Kb. The
resulting plasmid, NSPI-IL-31-mIgG-myc-His, was checked by
restriction mapping and sequencing. Plasmids were transfected into
Chinese hamster ovary (CHO) cells using FuGENEV R 6 (Roche,
Penzberg, Germany) in accordance with the manufacturer's
instructions. After 48 hours, cells were incubated in growth medium
containing puromycin (1 .mu.l/ml) to select for stable
transfectants. Conditioned medium from CHO transfected cells were
tested for inhibition of cell viability.
Statistical Analysis
[0150] Data are presented as mean standard deviation (SD).
Statistically significant differences were determined by two-tailed
Student's t test or one-way ANOVA as described in the text.
Significance was set at values of *, 0.05>p>0.01; **,
0.01>p>0.001; and ***p<0.001.
Example 1
IL-31 Affects Tumor Cell Proliferation and Viability
[0151] Cell lysates from tumor cell lines including EMT6, P3, MPC,
CT26, B16, LLC, K7M2, 4T1 and MC38 were analyzed for IL-31 as well
as IL-31 receptor (IL-31R) expression using western blot.
[0152] As detailed herein and as presented in FIG. 1(A-G), IL-31
inhibits tumor cell proliferation in IL-31 dependent manner FIG. 1A
shows cell lysates from tumor cell lines that were analyzed for
IL-31 as well as IL-30 receptor (IL-31R) expression using western
blot analysis. Particularly, the results in FIG. 1A show that while
IL-31 was highly expressed in MC38 and K7M2 cell lines, it was
minimally expressed in P3, MPC, B16 and 4T1 cells. In addition,
IL31R shows high expression patterns in most non-metastatic cells
when compared to highly metastatic cells which shows low expression
pattern.
[0153] Next, MC38 and 4T1 cell lines were used as two
representative cells which either express IL-31 or not,
respectively, in order to further evaluate the possible effect of
IL-31 on tumor cell proliferation and viability. Cell viability was
assessed in the presence of recombinant IL-31 (rIL-31) by Alamar
Blue. The results in FIGS. 1B-E demonstrates that the addition of
rIL-31 strongly and significantly inhibited cell proliferation in
MC38, CT26 and HCT116 when compared to control, an effect which was
absent in the case of 4T1 cells (FIG. 1C). In addition, cell count
was performed on these cells, and revealed that the number of
viable cells was significantly reduced in MC38 in the presence of
IL-31, but not in 4T1 tumor cells cultured with IL-31 (FIGS. 1F and
1G). These results suggest that IL-31 affects cell viability by
inhibiting cell proliferation but only in some tumor cell lines,
suggesting a distinct dependency of tumor cells on IL-31 and/or
IL-31R expression.
Example 2
Lack of IL-31 in Tumor Cells Promotes Tumor Growth and
Angiogenesis
[0154] To further confirm the activity of IL-31 on tumor cells in
vivo, IL-31 was silenced in MC38 cells using RNAi technique.
[0155] As related to in detail herein, FIG. 2 presents shRNA for
IL-31 inhibits the expression of IL-31 in tumor cells. FIG. 2A
shows lysates of MC38 murine colon carcinoma cells after they were
stably transfected with scramble plasmid or plasmid containing
shRNA for IL-31 were evaluated for IL-31 expression using western
blot analysis. The graph in FIG. 2B shows the percentage of
reduction in IL31 expression in MC38 shIL-31 after it was
normalized to its expression in scrambled control MC-38, as
assessed by densitometry.
[0156] As can be seen in FIG. 2B, the expression level of IL-31 in
MC38 shIL-31 cells was reduced by more than 60% compared to
scrambled MC38 control cells.
[0157] As further detailed herein, FIG. 3 presents the lack of
IL-31 expression promotes tumor growth, angiogenesis and TAMs. Two
million scramble control MC38, or its shIL-31 MC38 counterparts
cells were injected subcutaneously into the flanks of C57Bl/6 mice.
In FIG. 3A tumor growth was assessed regularly using a caliper. At
end point, tumors were removed, sectioned and then immunostained
with CD31, as an endothelial cell marker (FIG. 3B). Nuclear
staining is designated by DAPI. In a parallel experiment, tumors
were removed at end point and prepared as single cell suspension
for the evaluation of endothelial cells (CD31+ cells) and
macrophages (F4/80+ cells) using flow cytometry. The percentage of
(C) CD31+ cells and (3D) F4/80+ cells are presented. **,
0.05<p<0.01 (FIGS. 3 C-D).
[0158] The results in FIG. 3A show that tumor growth was
significantly enhanced in MC38 shIL-31 tumors when compared to
their scrambled MC-38 counterparts. At the end point, tumors were
removed and analyzed for microvessel density (MVD). A significant
increase in MVD was observed in tumors from MC38 siIL-31 cells when
compared to control tumors (FIG. 3B). In addition, large vessel
structures were detected in tumors from MC38 shIL-31 tumors when
compared to MC38 control counterparts (FIG. 3B). These results were
further confirmed when the tumors were prepared as single cell
suspension and analyzed by flow cytometry to CD31.
[0159] Recent studies indicated that bone marrow derived cells
(BMDC) colonizing tumors may affect angiogenesis. Tumor associated
macrophages (TAMs), for example, have been found to contribute
significantly to tumor angiogenesis and subsequently promote tumor
growth. Accordingly, macrophage colonization of tumors from MC38
shIL-31 cells was compared to control tumors. The number of
macrophages (F4/80+ cells) and endothelial cells (CD31+ cells) was
significantly higher in shIL-31 tumors when compared to
ev-scrambled control tumors (FIGS. 3C and 3D). Taken together,
these results suggest that the lack of IL-31 in tumors that
previously expressed IL-31 promoted tumor growth, increased
angiogenesis, and supported macrophage colonization of tumors.
Example 3
IL-31 Induces Anti-Tumor Activity in Both MC38 and 4T1 Tumors
[0160] As detailed herein, FIG. 4 present inhibition of tumor
growth, angiogenesis, and metastasis by IL-31. Two million of MC38
cells were implanted into the flanks of C57Bl/6 mice and half a
million 4T1 cells were implanted into the mammary fad pad of BALB/c
mice and were left to grow until they reached 150-200 mm.sup.3, at
which point the mice were implanted with minipumps containing PBS
(control) or 0.7 .mu.g/day recombinant IL-31 (rIL-31). Tumor growth
was assessed regularly for (FIG. 4A) MC-38 and (FIG. 4B) 4T1. To
test whether adding recombinant IL-31 can inhibit the growth of
tumors, both MC38 cells, which are known to express IL-31, as well
as 4T1 cells, which do not express IL-31, were used. To this end,
two million MC38 cells were subcutaneously implanted into the
flanks of C57/Black mice (n=5/group) and half a million 4T1 cells
were orthotopically implanted into the mammary fad pad of BALB/c
mice (n=5/group). When tumors reached a size of .about.150
mm.sup.3, recombinant mouse IL-31 was continuously infused into the
mice using osmotic pumps as explained above (n=5/group). The
subcutaneously implanted osmotic minipumps were loaded with
recombinant IL-31 that was released at a dose of 0.7 .mu.g per day.
Minipumps loaded with vehicle control (PBS) were used in control
mice (n=5). Tumor growth was assessed regularly or at end point. In
both mice groups, the continuous infusion of IL-31 resulted in a
significant reduction in tumor size when compared to control tumors
(FIGS. 4A and 4B). In addition, at end point, MC38 tumors were
removed, sectioned and then (FIG. 4C) immunostained with CD31, as
an endothelial cell marker. Nuclear staining is designated by DAPI.
FIG. 4D shows quantification of the number of microvessels is
presented in the graph. When MC38 tumors were removed and assessed
for microvessel density (MVD), a decreased MVD was observed in
tumors from mice that underwent IL-31 infusion when compared to
control those injected with vehicle control (see in FIGS. 4C and
4D). Importantly, since the 4T1 tumor model is known to
aggressively metastasize to the lungs of mice, at end point, tumors
from all groups (n=5 mice/group) were removed and lungs were
analyzed for metastatic lesions. In the case of 4T1 tumors, at end
point, lungs were removed from the mice and assessed for metastatic
lesions using H&E staining of lung sections (FIG. 4E). As
depicted in FIG. 4E, a significant lower number of lung metastatic
lesions were observed in mice infused with recombinant IL-31 when
compared to lungs from mice infused with PBS. Collectively, these
results suggest that the continuous infusion of mouse IL-31 has a
numerous anti-tumor activities on tumor and its metastatic
sites.
(n FIG. 4F quantification of the number of metastatic lesions per
field is provided. ***, p<0.001. In FIG. 4G NOD-SCID mice were
implanted with HCT116 human colon carcinoma cells (2.times.10.sup.6
cells; n=5 mice/group). When the tumors reached a size of 50
mm.sup.3, the mice were either implanted with pumps containing 150
.mu.g hIL31-IgG protein or injected ip twice a week with 50 .mu.g
hIL-31-IgG. Tumor growth was assessed over time. The results show
that both treatment methodologies resulted in a significant
inhibition in tumor growth. After 2 weeks of treatment tumors were
removed and sectioned. Tumor sections were stained for CD31, an
endothelial cell marker, and the microvessel density was evaluated,
by counting the number of vessels per field. As shown in the
figure, the number of microvessels in the treated tumors was
significantly lower than their numbers in control tumors. (4H-I)
HCT116 tumors were removed at the end point, after mice were
treated with hIL-31-IgG for 2 weeks either by pump or by IP
injections. Tumors were sectioned and stained for CD31 (an
endothelial cell marker--in red) FIG. 4H. Quantification of the
number of vessels--microvessel density (MVD) per field is provided
in FIG. 4I. As seen in 4H-I, tumors from mice treated with IL31-IgG
(either by injection or in pump) have lower microvessel density
than control tumors, suggesting an anti-angiogenic activity of
IL-31.
Example 4
Macrophages are Skews Towards M1 Phenotype in the Presence of
IL-31
[0161] The in vivo results further suggested that IL-31, in
addition to its effects on the tumor vasculature and the inhibition
of tumor cell viability, as shown in vitro, may act also as a
factor that can alter the macrophage colonization of tumors. In
order to assess whether IL-31 may alter the phenotype properties of
macrophages, an experiment focused on two specific macrophage
phenotypes, known as pro-inflammatory M1 (CD206-/CD11c+) phenotype
and anti-inflammatory M2 (CD206+/CD11c-) phenotype, was conducted.
To this end, J774 murine macrophage cell lines were cultured in the
presence or absence of recombinant IL-31 for 48 hours.
Subsequently, they were analyzed by flow cytometry for the
expression of M1 and M2 macrophages.
[0162] As detailed herein, FIG. 5 presents: IL-31 promotes
macrophage polarization into M1 phenotype. (A) J774 murine
macrophages cell lines were cultured in serum-free medium in the
presence or absence of 100 ng recombinant IL-31 for 24 hours. After
48 hours, cells were immunostained with F4/80, CD206, and CD11c to
evaluate the percentage of M1 (CD11c+/CD206-) and M2
(CD11c-/CD206+) macrophages. Graphs are provided. (B) MC38 tumors
implanted in C57B16 which, either express IL-31 (ev-scrambled) or
not (shIL-31), were let to grow until endpoint. Tumors were then
removed and prepared as single cell suspension, and the percentage
of M1 and M2 macrophages colonizing tumors were analyzed using flow
cytometry. (C) Two million MC38 cells were implanted in the flanks
of C57Bl/6 mice. When tumors reached 150-200 mm.sup.3 mice were
implanted with mini-pumps containing PBS (control) or recombinant
IL-31 in a dose of 0.7 .mu.g/day (rIL-31). At end point, tumors
were removed and prepared as single cell suspension. The percentage
of M1 and M2 macrophages colonizing tumors were analyzed using flow
cytometry. **, 0.05<p<0.01; ***, p<0.001.
[0163] The results in FIG. 5A revealed a significant increase in M1
phenotype and a decrease in M2 phenotype in J774 cells cultured in
the presence of IL-31 when compared to control cells.
[0164] Next, the colonization of macrophages in tumors was
re-examined in mice bearing MC-38 tumors (n=5/group), which either
do not express IL-31 (shIL31 tumors) or that were implanted in mice
infused with recombinant IL-31 (n=5/group), both compared to
control mice (n=5). The results in FIG. 5B show a reversed
phenotype of M1 and M2 macrophages colonizing shIL-31 and control
tumors, namely, the percentage of M1 macrophages was significantly
reduced and the percentage of M2 macrophages was significantly
increased in the microenvironment of tumors lacking IL-31
expression (shIL-31) when compared to control tumors. In addition,
tumors from mice infused with recombinant IL-31 revealed that the
percentage of M2 macrophages but not M1 macrophages colonizing MC38
tumors was significantly reduced in mice infused with recombinant
IL-31 when compared to mice infused with vehicle control (FIG. 5C).
Taken together, the results further suggest that both in vivo and
in vitro, IL-31 reduces the anti-inflammatory macrophage phenotype
in tumors.
Example 5
IL-31-IgG Construct Inhibits the Proliferation of Tumor Cells
[0165] Since the continuous infusion of IL-31 revealed anti-tumor
activities a construct in which IL-31 was conjugated to
immunoglobulin in order to increase the half-life of conjugated
cytokine was assessed. IL-31-IgG was prepared by cloning a plasmid
containing IL-31 conjugated with Mouse IgG1 heavy chain
(hinge-CH2-CH3) to give a product of IL-31-mIgG-myc-His. The
plasmid was transfected into Chinese hamster ovary (CHO) cells.
Conditioned medium from transfected CHO cells was then placed on
MC38 cells and then tested for activity using Alamar Blue assay as
detailed in Example 1.
[0166] As detailed herein, FIG. 6 presents MC38 cell viability that
was assessed using Alamar Blue assay. The cells were cultured in
the presence of escalating doses of mIL-31-IgG. A reduction in cell
viability was observed with the increased concentration of
IL-31-IgG.
Example 6
Tumors can be Affected by the Treatment with the IL-31 Ligand,
which Inhibits Tumor Cell Viability
[0167] The expression levels of IL-31R (receptor) on various human
colon and breast carcinomas from cancer patients revealed that many
but not all tumors express IL-31R. Thus, such tumors can be
affected by the treatment with the ligand IL-31 which inhibits
tumor cell viability as shown in vitro.
[0168] Table 1 is a summary of biopsies from colon and breast
cancer patients that were immunostained for IL-31R. The table show
that most biopsies have positive (P) staining for IL-31R (N-is
referred to negative staining). Intensity of staining is presented
by the number of "+" signs in each sample. FIG. 7 provides an
example of staining of one of the biopsies, indicating the high
expression of IL-31R (in black) in carcinogenous tissue. Table 2
provides a tissue array of human normal and tumor tissue of various
origins, indicating the expression levels of IL-31R in each tissue.
High expression of IL31R is presented in carcinogenous tissues of
all origins. However, in some normal tissue high expression of
IL31R is also presented. "T" stands for tumor tissue. "N" stands
for normal tissue.
TABLE-US-00007 TABLE 1 biopsies from colon and breast cancer
patients that were immunostained for IL-31R Tissue type/Patient
number Staining (P/N) Colon Cancer Biopsies 12/15 Positives
Staining Intensity 14-09085 P ++ 14-07461 P ++++ 14-05245 P ++
14-05087 P ++ 14-04018 P ++++ 14-03717 N - 14-02841 P + 14-01064 N
- 14-00670 P ++ 14-00154 P ++ 14-09531 P ++++ 14-09428 N + 14-09434
P +++ 14-09431 P ++++ 14-09087 P +++ Breast Cancer 6/10 positives
Staining Intensity 68482/10 N - 19197/11 P + 14994/14 N - 32530/10
N - 5378/10 P ++ 8603/13 N - 22782/14 P ++ 49549/12 P +++ 29788/11
P +++ 9757/13 P + Breast Cancer Biopsies 4/5 positives Staining
Intensity 34735/12 P ++++ 48685/09 P +++ 28176/10 P ++ 1637/10 N -
12855/11 P +++
TABLE-US-00008 TABLE 2 IL-31R in human normal and tumor tissue of
various origins Cancer Survey Tissue Array Tissue type Tumor (T)
Normal (N) Remark Breast 4/6 1/2 Most T are mild Colon 6/7 2/3 Most
T are mild Lung 8/8 3/3 Different intensities in T mild stain in N
Kidney 8/8 5/5 N is also strong Ovary 8/9 1/4 Different intensity
in T Endometrium 5/5 2/3 T are intense Stomach 5/8* 0/0 *2 T -cores
are connective tissue Skin 6/6 1/2 Liver 5/6* 5/5 *1 T core is
connective tissue
[0169] The expression levels of IL-31R on various human colon and
breast carcinomas from cancer patients revealed that many but not
all tumors express IL-31R. Thus, such tumors can be affected by the
treatment with the ligand IL-31 which inhibits tumor cell viability
as shown in vitro.
Example 7
IL-31-IgG is Stabilized for at Least 72 h in Peripheral Blood
[0170] The IL-31-IgG protein stabilization was tested by injecting
C57B16 mice 30 .mu.g of the indicated IL-31 proteins (both human
and mice). Blood was drawn by retro-orbital sinus at different time
points, and plasma was separated. Plasma (2 .mu.l) was used to
detect the various IL-31 proteins using anti-His HRP conjugated
antibody by Western Blot. From FIGS. 9G-9I it can be seen that the
different IL-31-IgG proteins are stabilized for at least 72 h in
peripheral blood. It is further noted that FIG. 8 shows that
IL-31-IgG is stabilized for at least 72 hours in peripheral blood.
293T cells were transfected with the IL-31-Ig construct (FIG. 8A).
Conditioned medium (CM) and lysates were obtained after 48 hours
and were then detected for the various components of the IL-31
construct by Western Blot. (FIG. 8B-E). Detection of the miL-31
part (FIG. 8B), the mCH2-CH3-IgG part (FIG. 8C), the Myc part (FIG.
8D) and the His part (FIG. 8E) using the Goat-a-Rat, G-a-m-IgG,
G-a-m-light, G-a-m-light antibodies, respectively. Coomassie
Brilliant Blue (CBB) (stain of 20 mg purified hIL-31-IgG and
mIL-31-IgG is shown in FIG. 8 F. The protein IL-31-IgG (both human
and mouse), which was generated and purified, has been tested for
its stability in peripheral blood of mice. C57B16 mice were
injected with 30 .mu.g of the indicated IL-31 proteins. Blood was
drawn by retro-orbital sinus at different time points, and plasma
was separated. Plasma (2 .mu.l) was used to detect the various
IL-31 proteins using anti-His HRP conjugated antibody by Western
Blot. (FIG. 9G) 30 .mu.g mIL31-IgG Vs. 200 mIL31. (FIG. 8H) 30
.mu.g hIL31-IgG Vs. 200 .mu.g hIL31. (FIG. 9I) 30 .mu.g hIL31-IgG
Vs. 200 .mu.g hIL31-PEG. hIL-31-PEG represents purified IL-31
protein following PEGylation. FIG. 8G-H represent the
pharmacokinetics of IL31-IgG in the peripheral blood of mice. It
shows that while IL-31 is in its native form, it is cleared from
the system within 15 min, IL31-IgG is stably present in peripheral
blood for the first 72 hours.
Example 8
IL-31 Directly Inhibits Angiogenesis
[0171] IL-31 directly inhibits angiogenesis as assessed by tube
forming assay:
[0172] Human umbilical vascular endothelial cells (HUVECs) were
seeded in Matrigel-coated 48-well tissue culture plates
(4.times.104 cells/well) and incubated in 20% 1-BS M-199 medium.
Wells were cultured with 100 ng/ml recombinant human IL-31 or 10
ug/ml human IL-31-IgG. The cells were cultured and phase-contrast
images of microvessel tubes were captured after 200 min at
100.times. magnification using the Leica CTR 6000 (Leica
Microsystems). The images were analyzed using Image) software and
quantified by counting the number of HUVEC junctions (bifurcations)
per field.
[0173] FIG. 9A shows representative images of tube forming of
HUVECs in the presence of 100 ng/ml recombinant human IL-31
(rhIL31) or 10 .mu.g/ml IL-31-IgG are provided 200 min time-point.
The number of bifurcations per field were quantified and presented.
*, p<0.05; ***, p<0.001 as shown in FIG. 9B. As can be seen,
IL31 or IL31IgG significantly inhibits tube forming.
[0174] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
Sequence CWU 1
1
121164PRTHomo sapiens 1Met Ala Ser His Ser Gly Pro Ser Thr Ser Val
Leu Phe Leu Phe Cys 1 5 10 15 Cys Leu Gly Gly Trp Leu Ala Ser His
Thr Leu Pro Val Arg Leu Leu 20 25 30 Arg Pro Ser Asp Asp Val Gln
Lys Ile Val Glu Glu Leu Gln Ser Leu 35 40 45 Ser Lys Met Leu Leu
Lys Asp Val Glu Glu Glu Lys Gly Val Leu Val 50 55 60 Ser Gln Asn
Tyr Thr Leu Pro Cys Leu Ser Pro Asp Ala Gln Pro Pro 65 70 75 80 Asn
Asn Ile His Ser Pro Ala Ile Arg Ala Tyr Leu Lys Thr Ile Arg 85 90
95 Gln Leu Asp Asn Lys Ser Val Ile Asp Glu Ile Ile Glu His Leu Asp
100 105 110 Lys Leu Ile Phe Gln Asp Ala Pro Glu Thr Asn Ile Ser Val
Pro Thr 115 120 125 Asp Thr His Glu Cys Lys Arg Phe Ile Leu Thr Ile
Ser Gln Gln Phe 130 135 140 Ser Glu Cys Met Asp Leu Ala Leu Lys Ser
Leu Thr Ser Gly Ala Gln 145 150 155 160 Gln Ala Thr Thr 2495DNAHomo
sapiens 2atggcctctc actcaggccc ctcgacgtct gtgctctttc tgttctgctg
cctgggaggc 60tggctggcct cccacacgtt gcccgtccgt ttactacgac caagtgatga
tgtacagaaa 120atagtcgagg aattacagtc cctctcgaag atgcttttga
aagatgtgga ggaagagaag 180ggcgtgctcg tgtcccagaa ttacacgctg
ccgtgtctca gccctgacgc ccagccgcca 240aacaacatcc acagcccagc
catccgggca tatctcaaga caatcagaca gctagacaac 300aaatctgtta
ttgatgagat catagagcac ctcgacaaac tcatatttca agatgcacca
360gaaacaaaca tttctgtgcc aacagacacc catgaatgta aacgcttcat
cctgactatt 420tctcaacagt tttcagagtg catggacctc gcactaaaat
cattgacctc tggagcccaa 480caggccacca cttaa 4953492DNAMus musculus
3atgatcttcc acacaggaac aacgaagcct accctggtgc tgctttgctg tataggaacc
60tggctggcca cctgcagctt gtccttcggt gccccaatat cgaaggaaga cttaagaact
120acaattgacc tcttgaaaca agagtctcag gatctttata acaactatag
cataaagcag 180gcatctggga tgtcagcaga cgaatcaata cagctgccgt
gtttcagcct ggaccgggaa 240gcattaacca acatctcggt catcatagca
catctggaga aagtcaaagt gttgagcgag 300aacacagtag atacttcttg
ggtgataaga tggctaacaa acatcagctg tttcaaccca 360ctgaatttaa
acatttctgt gcctggaaat actgatgaat cctatgattg taaagtgttc
420gtgcttacgg ttttaaagca gttctcaaac tgcatggcag aactgcaggc
taaggacaat 480actacatgct ga 4924681DNAMus musculus 4gtgcccaggg
attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc 60ttcatcttcc
ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg
120tgtgttgtgg tagacatcag caaggatgat cccgaggtcc agttcagctg
gtttgtagat 180gatgtggagg tgcacacagc tcagacgcaa ccccgggagg
agcagttcaa cagcactttc 240cgctcagtca gtgaacttcc catcatgcac
caggactggc tcaatggcaa ggagttcaaa 300tgcagggtca acagtgcagc
tttccctgcc cccatcgaga aaaccatctc caaaaccaaa 360ggcagaccga
aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag
420gataaagtca gtctgacctg catgataaca gacttcttcc ctgaagacat
tactgtggag 480tggcagtgga atgggcagcc agcggagaac tacaagaaca
ctcagcccat catggacaca 540gatggctctt acttcgtcta cagcaagctc
aatgtgcaga agagcaactg ggaggcagga 600aatactttca cctgctctgt
gttacatgag ggcctgcaca accaccatac tgagaagagc 660ctctcccact
ctcctggtaa a 6815696DNAHomo sapiens 5gagcccaaat cttgtgacaa
aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60gggggaccgt cagtcttcct
cttcccccca aaacccaagg acaccctcat gatctcccgg 120acccctgagg
tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
180aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg
ggaggagcag 240tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc
tgcaccagga ctggctgaat 300ggcaaggagt acaagtgcaa ggtctccaac
aaagccctcc cagcccccat cgagaaaacc 360atctccaaag ccaaagggca
gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420gaggagatga
ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
480gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa
gaccacgcct 540cccgtgctgg actccgacgg ctccttcttc ctctacagca
agctcaccgt ggacaagagc 600aggtggcagc aggggaacgt cttctcatgc
tccgtgatgc atgaggctct gcacaaccac 660tacacgcaga agagcctctc
cctgtctccg ggtaaa 6966163PRTMus musculus 6Met Ile Phe His Thr Gly
Thr Thr Lys Pro Thr Leu Val Leu Leu Cys 1 5 10 15 Cys Ile Gly Thr
Trp Leu Ala Thr Cys Ser Leu Ser Phe Gly Ala Pro 20 25 30 Ile Ser
Lys Glu Asp Leu Arg Thr Thr Ile Asp Leu Leu Lys Gln Glu 35 40 45
Ser Gln Asp Leu Tyr Asn Asn Tyr Ser Ile Lys Gln Ala Ser Gly Met 50
55 60 Ser Ala Asp Glu Ser Ile Gln Leu Pro Cys Phe Ser Leu Asp Arg
Glu 65 70 75 80 Ala Leu Thr Asn Ile Ser Val Ile Ile Ala His Leu Glu
Lys Val Lys 85 90 95 Val Leu Ser Glu Asn Thr Val Asp Thr Ser Trp
Val Ile Arg Trp Leu 100 105 110 Thr Asn Ile Ser Cys Phe Asn Pro Leu
Asn Leu Asn Ile Ser Val Pro 115 120 125 Gly Asn Thr Asp Glu Ser Tyr
Asp Cys Lys Val Phe Val Leu Thr Val 130 135 140 Leu Lys Gln Phe Ser
Asn Cys Met Ala Glu Leu Gln Ala Lys Asp Asn 145 150 155 160 Thr Thr
Cys 78PRTHomo sapiens 7Gly Pro Leu Gly Ile Ala Gly Gln 1 5
85PRTHomo sapiensmisc_feature(5)..(5)Xaa is
epsilon-maleimidocaproic 8Lys Lys Phe Ala Xaa 1 5 98PRTHomo
sapiensmisc_feature(5)..(5)Xaa is D-arginine 9Lys Gly Ala Ser Xaa
Phe Thr Gly 1 5 109PRTHomo sapiensmisc_feature(1)..(1)Xaa is
epsilon-maleimidocaproic 10Xaa Arg Arg Ser Ser Tyr Tyr Ser Gly 1 5
1131DNAArtificial Sequenceprimer 11taccgctcga ggtgcccagg gattgtggtt
g 311225DNAArtificial Sequenceprimer 12cgttcgaatt taccaggaga gtggg
25
* * * * *
References