Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
TREATING OR PREVENTING CANCERS OVER-EXPRESSING REG4 OR KIAA0101
The present application claims the benefit of U.S. Provisional Application No.
60/838,649, filed August 18, 2006, and U.S. Provisional Application No.
60/838,749, filed
August 18, 2006, the entire disclosures of each of which are hereby
incorporated herein by
reference for all purposes.
TECHNICAL FIELD
The present invention relates to the field of biological science, more
specifically to the
field of cancer research. In particular, the present invention also relates to
methods of
treating and preventing cancer, for example pancreatic cancer, prostatic
cancer, breast cancer,
and bladder cancer. In particular, the present invention relates a
coinposition comprising a
nucleic acid capable of inhibiting expression of the gene encoding REG4 and
KIAA0101. In
some embodiments, the coinpound is a small interfering RNA (siRNA)
corresponding to a
subsequence from these genes.
Alternatively, the present invention also relates to methods of treating or
preventing
pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), in a
subject
comprising the step of administering to said subject a pharmaceutically
effective amount of an
antibody or fragment thereof that binds to a protein encoded by REG4.
Moreover, the
present invention relates a composition comprising a peptide of inhibiting the
interaction of
ICAA0141 with PCNA. In some embodiments, the compound is a cell-permeable
dominant-negative peptides have conserved PCNA-binding motif (PIP box).
BACKGROUND ART
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer
death in the western world and shows the worst mortality among the common
malignancies,
with a 5-year survival rate of only 4% (DiMagno EP, et al. Gastroenterology
1999; 117:
1464-84., Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.). In 2006, it
is estimated
that approximately 33,730 new cases are diagnosed to have pancreatic cancer in
the United
States and 32,300 of them are likely to die of the disease (Jemal A, et al.
Cancer statistics,
2006. CA Cancer J Clin 2006; 56: 106-130.). Since the majority of PDAC
patients are
diagnosed at their advanced stage, no effective therapy to cure the disease is
available at
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present, Several approaches in a combination of surgery with chemotherapy,
including 5-FU
or gemcitabine, with or without radiation, can improve patients' quality of
life (DiMagno EP,
et al. Gastroenterology 1999; 117: 1464-84., Wray CJ, et al. Gastroenterology
2005; 128:
1626-41.), but those treatments have a very limited effect on long-term
survival of PDAC
patients due to its extreniely aggressive and chemo-resistant nature.
The very poor prognosis of PDAC arises from several reasons that include the
difficulty of detection of PDACs at an early stage (DiMagno EP, et al.
Gastroenterology
1999; 117: 1464-84., Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.).
Despite
improvements in diagnostic imaging techniques such as endoscopic ultrasound
("EUS") or
magnetic resonance cholangiopancreaticography ("MRCP") (DiMagno EP, et al.
Gastroenterology 1999; 117: 1464-84., Wray CJ, et al. Gastroenterology 2005;
128: 1626-41.),
most patients do not undergo imaging procedures because they do not have any
symptoms
until late in the course of the disease. An accurate and easy serological
test, such as PSA
(prostate-specific antigen) for prostate cancer, could facilitate detection of
PDACs at an early
stage and can be applied for mass-screening of PDACs. Surgical resection of
early-staged
PDACs can offer the relatively favorable prognosis as 50-60% of five-year
survival (Wray CJ,
et al. Gastroenterology 2005; 128: 1626-41.). Hence, considering biological
aggressiveness
and resistance to chemotherapy of PDACs, one of the most realistic strategies
to improve the
prognosis of this fatal disease is to screen PDACs at an early stage by a non-
invasive
serological test.
Currently, CA19-9 is the only commercially available serological marlcer for
PDACs, but it is far from an ideal tumor marker, because (i) approximately 10-
15% of
individuals do not secrete CA19-9 due to their Lewis antigen status, (ii) it
is not specific to
pancreatic cancer and is also elevated in benign conditions, and (iii) it is
usually within a
normal range in patients at an early stage (Sawabu N, et al. Pancreas 2004;
28: 263-7.,
Pleskow DK, et al. Ann Intern Med 1989; 110: 704-9.). Hence, establishment of
a screeniiig
strategy thorough development of a novel tumor maker that is more specific and
more
sensitive to PDACs is urgently required.
REG4 has been reported to be a new member of the REG family (Hartupee JC, et
al. Biochim Biopliys Acta 2001; 1518: 287-93.), and as a tumor marker of PDAC.
The
molecules belonging to the REG (regenerating islet-derived) family are
secreted proteins
playing a role in tissue regeneration and inflammation in digestive organs
(Hartupee JC, et al.
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Biochim Biophys Acta 2001; 1518: 287-93., Watanabe T, et al. J Biol Chem 1990;
265:
7432-9., Uno M, et al. Adv Exp Med Biol 1992; 321: 61-6.). The expression
levels of the
members were reported to be up-regulated in several gastrointestinal cancers
and to function
as a trophic or anti-apoptotic factor in cancers (Unno M, et al. Adv Exp Med
Biol 1992; 321:
61-6., Sekikawa A, et al. Gastroenterology 2005; 128: 642-53.).
cDNA microarray technologies have enabled practitioners to obtain
comprehensive
profiles of gene expression in normal and malignant cells, and coinpare the
gene expression in
malignant and corresponding normal cells (Okabe et al., (2001) Cancer Res
61:2129-37;
Kitahara et al., (2001) Cancer Res 61: 3544-9; Lin et al., (2002) Oncogene
21:4120-8;
Hasegawa et al., (2002) Cancer Res 62:7012-7). This approach enables the
disclosure of the
complex nature of cancer cells, and helps to understand the mechanism of
carcinogenesis.
Identification of genes that are deregulated in tumors will lead to more
precise and accurate
diagnosis of individual cancers, and to develop novel therapeutic targets
(Bienz and Clevers,
(2000) Cell 103:311-20).
For example, recent years, a new approach of cancer therapy using gene-
specific
siRNA was attempted in clinical trials (Bumcrot D et al., Nat Chem Bio12006
Dec, 2(12):
711-9). RNAi seems to have already earned a place among the major technology
platforms
(Putral LN et al., Drug News Perspect 2006 Jul-Aug, 19(6): 317-24; Frantz S,
Nat Rev Drug
Discov 2006 Jul, 5(7): 528-9; Dykxhoorn DM et al., Gene Ther 2006 Mar, 13(6):
541-52).
Nevertheless, there are several challenges that need to be faced before RNAi
can be applied in
clinical use. These challenges include overcoming poor stability of RNA in
vivo (Hall AH et
al., Nucleic Acids Res 2004 Nov 15, 32(20): 5991-6000, Print 2004; Amarzguioui
M et al.,
Nucleic Acids Res 2003 Jan 15, 31(2): 589-95), reducing the toxicity as an
agent (Frantz S,
Nat Rev Drug Discov 2006 Jul, 5(7): 528-9) and selecting the suitable mode of
delivery, the
precise sequence of the siRNA or shRNA used, and cell type specificity. It is
well-known
fact that there are possible toxicities related to non-specific silencing
because of partial
homology, or induction of the interferon response by inducing double-stranded
molecules
(Judge AD et al., Nat Biotechno12005 Apr, 23(4): 457-62, Epub 2005 Mar 20;
Jackson AL &
Linsley PS, Trends Genet 2004 Nov, 20(11): 521-4). So, double-stranded
molecules
targeting cancer-specific genes must be improved to be devoid of adverse
effects.
Earlier the present inventors performed detailed and accurate expression
profile
analysis of pancreatic cancers using a genome-wide cDNA microarray consisting
of
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approximately 27,000 genes, in combination with laser microdissection to
purify cancer cell
population (Nakamura et al., (2004) Oncogene, 23: 2385-400). Among the genes
the present
inventors identified as being trans-activated in pancreatic cancer cells, the
present inventors
here focused on KIAA0101 as a molecular target for cancer therapy (W02004/03
1412).
PCNA (proliferating cell nuclear antigen) is essential for DNA replication and
DNA repair as well as influencing cell cycle progression through interacting
with several cell
cycle proteins (Prelich et al., (1987) Nature, 326: 471-5; Wyman and Botchan
(1995) Curr
Biol, 5: 334-7; Warbrick et al., (2000) Bioessays, 22: 997-1006). The crystal
structure
showed that PCNA is a ring-shaped homotrimeric protein and functions as a
clamping
platform necessary to recruit to DNA proteins involved in DNA synthesis or
metabolism,
such as DNA polymerases, DNA ligase, and others (Krishna et al., (1994) Cell,
79: 1233-43).
PCNA interacts with numerous DNA replication/repair enzymes, and several
proteins have its
conserved PCNA-binding motif (PIP box, QXXL/I/M=/Y) through which they
interact
with PCNA (Jonsson et al., (1998) EMBO J, 17: 2412-25). Overexpression of PCNA
is a
hallmark of cell proliferation and in the clinic PCNA serves as a general
proliferative marker,
especially in the prognosis of tumor development as well as Ki67/ MIB-1
(Haitel et al., (1997)
Am J Clin Pathol, 107: 229-3 5).
KIAA0101 was previously identified as p15PAF (PCNA-associated factor) to bind
with PCNA protein by yeast two-hybrid screening (Yu et al., (2001) Oncogene
20: 484-9) and
it has the conserved PIP box through which it can interact with PCNA. However,
its
function remains unknown and how ICIAA0101-PCNA interaction can involve cell
proliferation or cancer progression is still a puzzle (Yu et al., (2001)
Oncogene, 20: 484-9;
Simpson et al., (2006) Exp Cell Res. 312: 73-85).
SUMMARY OF THE INVENTION
The present inventors here report over-expression of REG4, a new member of the
REG family, and/or KIAA0101 in cancer cells (e.g., in PDAC cells) on the basis
of the
genome-wide cDNA microarray analysis as well as RT-PCR and
imnlunohistochemical
analysis. Furthermore, the present inventors found that knockdown of the
endogenous
REG4 and/or K.IAA0101 expression in cancer (e.g., PDAC) cell lines with an
siRNA caused
3o drastic decrease of cell viability. Concordantly, addition of recombinant
REG4 and/or
KIAA.0101 to the culture medium enhanced growth of cancer (e.g., PDAC cell
lines in a dose-
dependent manner. A monoclonal antibody against REG4 and/or KIAA0101
neutralizes the
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growth-promoting effects and attenuated significantly the growth of cancer
(e.g., PDAC cells).
These findings implicate that REG4 and KIAA0101 are promising tumor markers to
screen
early-staged cancers, including PDAC, and also that neutralization of REG4
and/or
KIAA0101, for example, by the use of an antibody, an inhibitory polypeptide or
an inhibitory
polynucleotide (e.g., siRNA) offers an effective prophylactic or therapeutic
treatment against
cancers mediated by aberrant (i.e., abnormally high) REG4 and/or KIAA0101 over-
expression and/or intracellular signaling, including PDACs.
An objective of the present invention is to provide compounds useful in the
treatment and/or prevention of cancers mediated by aberrant (i.e., abnorinally
high) REG4
and/or KIAA0101 over-expression and/or intracellular signalling.
Alternatively, an
objective of the present invention is to provide pharmaceutical compositions
and methods for
either or both the treatment and prevention of such cancers, for example,
pancreatic cancer,
prostate cancer, breast cancer or bladder cancer using the compounds.
The present inventors coiifirm that suppression of REG4 or KIAA.O101
expression,
for example using an siRNA, can achieve the inhibition of cancer
proliferation. Accordingly,
it is an objective of the present invention is to provide method for treating
or preventing
pancreatic cancer in a subject comprising administering to said subject a
composition
comprising a small interfering RNA (siRNA) that inhibits expression of REG4.
In a further
embodiment, the invention provides methods for treating or preventing cancers
mediated by
aberrant (i.e., abnormally high) KIAA0101 over-expression and/or intracellular
signaling, for
example, pancreatic cancer, prostate cancer, breast cancer or bladder cancer
in a subject
comprising administering to said subject a composition comprising an siRNA
that inhibits
expression of KIAA0,101.
The present invention also provides a pharmaceutical composition for treating
or
preventing pancreatic cancer comprising a pharmaceutically effective amount of
a small
interfering RNA (siRNA) that inhibits expression of REG4 as an active
ingredient, and a
pharmaceutically acceptable carrier.. The present invention further provides a
pharmaceutical composition for treating or preventing cancers mediated by
aberrant (i.e.,
abnormally high) KIAA0101 over-expression and/or intracellular signaling, for
example,
pancreatic cancer, prostate cancer, breast cancer or bladder cancer
coniprising a
pharmaceutically effective amount of an siRNA that inhibits expression of
KIAA0101 as an
active ingredient, and a pharmaceutically acceptable carrier.
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The present invention further provides use of a small interfering RNA (siRNA)
that
inhibits expression of REG4 for manufacturing a pharmaceutical composition for
treating or
preventing pancreatic cancer, and use of a small interfering RNA (siRNA) that
inhibits
expression of KIAA0101 for manufacturing a pharmaceutical composition for
treating or
preventing cancers mediated by aberrant (i.e., abnormally high) KIAA0101 over-
expression
and/or intracellular signaling, for example, pancreatic cancer, prostate
cancer, breast cancer or
bladder cancer. In some embodiments, a preferable siRNA comprises the
nucleotide
sequence of SEQ ID NO: 5 or SEQ ID NO: 32 as the target sequence.
The present invention relates to a double-stranded molecule comprising a sense
strand and an antisense strand, wherein the sense strand comprises a
ribonucleotide sequence
corresponding to a target sequence of SEQ ID NO: 5 or SEQ ID NO: 32, and
wherein the
antisense strand comprises a ribonucleotide sequence which is complementary to
said sense
strand, wherein said sense strand and said antisense strand hybridize to each
other to form
said double-stranded molecule, and wherein said double-stranded molecule, when
introduced
into a cell expressing the REG4 gene or the KIAA0101 gene, inhibits expression
of said gene.
In addition, it is confirmed that REG4 functions as an autocrine or paracrine
growth
factor and mediate Alct signaling pathways. Furthermore, the present inventers
find that an
anti-REG4 antibody neutralizes the cell proliferative activity of the REG4 to
attenuate the
growth of pancreatic cancer cells.
Accordingly, it is an objective of the present invention is to provide a
method for
treating or preventing pancreatic cancer in a subject comprising administering
to said subject
an anti-REG4 antibody or fragment thereof that neutralizes REG4 activity. The
present
invention also provides a pharmaceutical composition for treating or
preventing pancreatic
cancer, said composition comprising a pharmaceutically effective amount of an
anti-REG4
antibody or fragment thereof that neutralizes REG4 activity as an active
ingredient, and a
pharmaceutically acceptable carrier. The present invention further provides
use of an anti-
REG4 antibody or fragment thereof that neutralizes REG4 activity for
manufacturing a
pharmaceutical composition for treating or preventing pancreatic cancer. In
some
embodiments, preferable anti-REG4 antibody is monoclonal antibody. In some
embodiments, anti-REG4 antibody comprises a VH and VL chain, each VH and VL
chain
comprising CDR amino acid sequences designated CDR1, CDR2 and CDR3 separated
by
framework amino acid sequences, the amino acid sequence of each CDR in each VH
and VL
chain is:
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VH CDRI : SYWIVIH (SEQ ID NO: 20),
VH CDR2 : NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3 : GGLWLRVDY (SEQ ID NO: 22),
VL CDRl : SASSSVSYMH (SEQ IDNO: 23),
VL CDR2 : DTSKLAS (SEQ ID NO: 24), and
VL CDR3 : QQWSSNPF (SEQ ID NO: 25).
The present invention also relates to methods for treatment and/or prevention
of
pancreatic cancer comprising the step of administering an antibody comprises a
VH and VL
chain, each VH and VL chain comprising CDR amino acid sequences designated
CDR1,
CDR2 and CDR3 separated by framework amino acid sequences, the amino acid
sequence of
each CDR in each VH and VL chain is:
VH CDRl : SYWMH (SEQ ID NO: 20),
VH CDR2 : NIIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3 : GGLWLRVDY (SEQ ID NO: 22),
VL CDR1 : SASSSVSYMH (SEQ IDNO: 23),
VL CDR2 : DTSKLAS (SEQ ID NO: 24), and
VL CDR3 : QQWSSNPF (SEQ ID NO: 25).
In addition, the present invention provides inhibitory polypeptides that
contain
QKGIGEFF (SEQ ID NO: 46). In some preferred embodiments, the amino acid
sequence is
VRPTPKWQKGIGEFFRLSPK (SEQ ID NO. 44) or TPKWQKGIGEFFRLSP (SEQ ID NO.
45). The present invention further provides pharmaceuticals or methods using
these
inhibitory polypeptides for prevention and/or treatment of cancer.
The present invention also relates to methods for treatment and/or prevention
of
cancer coinprising the step of administering an inhibitory polypeptide that
contains
QKGIGEFF (SEQ ID NO: 46), for example an inhibitory polypeptide having at
least a
fragment of the amino acid sequence VR.PTPKWQKGIGEFFRLSPK (SEQ ID NO: 44);
TPKWQKGIGEFFRLSP (SEQ ID NO. 45); or a polynucleotide encoding the same.
Furthermore, the present invention relates to the use of polypeptides of the
invention; or the
use of nucleotides encoding the same, in manufacturing pharmaceutical
formulations for the
treatment and/or prevention of cancer.
These and other objects and features of the invention will become more fully
apparent when the following detailed description is read in conjunction with
the
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accompanying figures and examples. However, it is to be understood that both
the foregoing
summary of the invention and the following detailed description are a
preferred embodiment,
and not restrictive of the invention or other alternate embodiments of the
invention.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 REG4 mRNA and protein expression levels in PDAC cells. (A) RT-PCR
analysis of REG4 and TUBA, as a quantitative control, in the microdissected
PDAC cells
(lanel-9) comparing with normal pancreatic ductal epithelial cells (NPD),
which were also
microdissected, normal pancreas, and normal vital organs including heart,
lung, liver, kidney
and brain. (B, C, D) In immunohistochemical study using anti-REG4 antibody,
intense
staining was observed in PDAC cells. Positive staining of REG4 was observed as
cytoplasmic granules (B), suggesting secretion of REG4, and at the cytoplasmic
membrane
(C). In normal pancreatic tissue, acinar cells showed very faint staining, but
not in normal
ductal epithelium cells and islet cells (D).
Fig. 2 Knockdown of REG4 expression by siRNA caused attenuation of
pancreatic cancer cell growth. (A) Knockdown effect on REG4 transcript was
validated by
semi-quantitative RT-PCR using cells transfected with an siRNA expressing
vector to REG4
(REG4-si2) and a negative control vector (siEGFP). 02-MG was used to quantify
RNAs.
REG4-si2 revealed strong knockdown effect, while EGFPsi did not show any
effect on the
level of REG4 transcript. (B, C) Transfection with a REG4-si2 vector into SUIT-
2 resulted
in drastic reduction of the numbers of viable cells measured by MTT assay (B)
and the
number of colony formation (C), compared with the cells transfected with
EGFPsi vectors
which did not showed any knockdown effect on REG4. Columns, average of
absorbance
from three experiments after a 7 day incubation with Geneticin; bars, SD. * p
< 0.01
(Student's t-test) at MTT assay (B).
Fig. 3 Growth-promoting effect of rhREG4 on PDAC cells. (A) The bioactive
rhREG4 proteins were generated by mammalian cells (FreeStyleTM 293). The
rhREG4 was
purified and analyzed by SDS-PAGE, followed by Coomassie staining (left) and
Western blot
(right) using specific antibody to REG4. (B) PK-45P cells were incubated with
0, 0.1, 1 and
lOnM rhREG4, supplied with 1%FBS. The treatment of rhREG4 stimulated cell
proliferation of PK-45P cells dose-dependently. Data point, average ratios of
absorbance
from three experiments compared with samples day 0; bars, SD. * p< 0.01
(Student's t test).
(C) Phosphorylation of Akt (Ser473) was enhanced dose-dependently by treating
PK-45P
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cells with 0, 0. l, 1 and lOnM rhREG4. Phosphorylated Akt was detected by
Western blot
using the antibody specific to phosphorylated Akt (Ser473), and the blots were
reprobedwith
antibody to Akt to evaluate the total level of Akt.
Fig. 4 Neutralizing and growth-suppressive effect of anti-REG4 monoclonal
antibody. (A) Binding affinity of anti-REG4 antibodies was evaluated by
immunoprecipitation using SUIT-2 culture medium, followed by Western blot
using anti-
REG4 pAb. Anti-REG4 mAb and pAb immunoprecipitated REG4 from SUIT-2 culture
medium with high affinity. (B) Anti-REG4 mAb treatment offset the growth-
promoting
effect of rhREG4. PK-45P was stimulated by l OnM rhREG4 in the presence or
absence of
anti-REG4 mAb. Columns, average ratios of absorbance from three experiments
compared
with samples grown in (-) medium; bars, SD. * p < 0.01 (Student's t tests).
(C) Effects of
various concentration of anti-REG4 mAb on the growth of SUIT-2 (REG4-positive)
and
MIAPaCa-2 (REG4-negative). Each cell line was incubated in the presence of
various
concentration of anti-REG4 mAb. Anti-REG4 mAb treatment suppressed SUIT-2 cell
growth dose-dependently while it did not affect the cell growth of MIAPaCa-2
that did not
express REG4 at all. Data point, average ratios of absorbance from three
experiments
compared with samples grown in (-) medium; bars, SD. * p < 0.01 (Student's t
tests). (D)
Anti-REG4 mAb treatment offset the phosphorylation of Alct that was stimulated
by rhREG4.
PK-45P cells were treated with lOnM rhREG4 in presence or absence of anti-REG4
mAb.
Phosphorylation of Akt was evaluated by Western blot using the antibody
specific to
phosphorylated Akt (Ser473), and the blots were reprobed with antibody to Akt
to evaluate
the total level of Akt. 1, non-stimulated; 2, l OnM rhREG4; 3, l OnM rhREG4 +
anti-REG4
mAb.
Fig. 5 Anti-REG4 antibody treatment suppressed pancreatic cancer cell growth
in
vivo. (A) Tumor volumes which were inoculated into nude mice were evaluated
during the
treatment of anti-REG4 antibody (34-1 mAb; n=8) with two times per a week (300
u
g/mouse i.p.) or control antibody (normal mouse IgG; n=9). The treatment of 34-
1 mAb
(shown in solid round marker and bold line) induced significant reduction of
tumor volumes
comparing to control IgG (P=0.0598). (B) Tumors were weiglited at 30 days
after first
treatment of anti-REG4 antibody (34-1 mAb) or control antibody. The treatment
of 34-1
niAb (shown in black box) induced significant reduction of tumor weights
comparing to
control antibody (P=0.0489).
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Fig. 6 REG4 over-expression contributed to y-ray resistance of pancreatic
cancer.
(A) pCAGGSnHC-REG4-HA was transfected to pancreatic cancer cell line PK-45P
that did
not express REG4. After the selection of Geneticin resistant cells, expression
of
recombinant REG4 at cell line was checked by western blotting assay. (B) After
48 hours
pre-incubation, REG4-expressing clones (C1-6, C2-6 and C10) or control clones
(M1, M3 and
M6) were y-irradiated at 1, 5, 10, or 30 Gyby using a 60Co source. After 48
hours, viable
cells were measured by using cell-counting, and relative ratio of absorbance-
(each
irradiation)/absorbance-(no-irradiation) was evaluated. (C) After 0, 1, or 5
Gy y-irradiation,
y ray-induced apoptosis was evaluated by detecting sub-G1 fraction using flow
cytometer.
Fig. 7 REG4 over-expression contributed to gemcitabine-resistance of
pancreatic
cancer. (A) After 48 hours pre-incubation, REG4-expressing clones (CI-6, C2-6,
C10) or
control clones (M1, M3, M6) were treated with 0.1-100,000 nM gemcitabine for
48 hours.
After incubation, viable cells were measured by using Cell-counting, and
relative ratio of
absorbance-(each treatment)/absorbance-(no treatment control) was evaluated,
(B) After
treating lOnM or 50nM gemcitabine for 48 hours, apoptosis was evaluated by
detecting sub-
Gl fraction using flow cytometer.
Fig. 8 The immunostaining of REG4 in the pancreatic adenocarcinoma specimens
undergoing neo-CRT. (A), (B) The pancreatic adenocarcinoma specimens
responding to
neo-adjuvant chemo-radiation therapy (neo-CRT) showed low or no expression of
REG4.
(C), (D) The pancreatic adenocarcinoma specimens non-responding to neo-CRT
showed
strong expression of REG4.
Fig. 9 The results of over-expression of KIAA0101 in pancreatic cancer cells.
(A) Semi-quantitative RT-PCR validated that KIAA0101 expression was up-
regulated in the
microdissected pancreatic cancer cells compared with normal pancreatic duct
cells which
were also microdissected and normal pancreatic tissue. Expression of TUBA
served as the
quantitative control. (B) Northern blot analysis showed the strong expression
of ICAA0101
in pancreatic cancer cell lines (lanes 1-6), while no expression was observed
in vital organs
including heart, lung, liver, kidney, and brain (lanes 7-11). (C)
Immunohistochemical study
using anti-KIAA0101 antibody. Intense staining was observed in the nuclei of
pancreatic
cancer cells (arrowhead x400), while acinar cells and normal ductal epithelium
in normal
pancreatic tissue showed no staining.
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Fig. 10 The result of effect of KIAA0101 knockdown by siRNAs on growth of
pancreatic cancer cells. (A) Two siRNA expression vectors specific to KIAA0101
transcript
(#759si) and an EGFP siRNA expression vector (EGFPsi) as a negative control
were
transfected into KLM-1 cells. Knockdown effect on KIAA0101 transcript was
validated by
RT-PCR, with P2MG expression as a quantitative control. Transfection with
#759si showed
strong knockdown effect, while EGFPsi did not show any effect on the level of
KIAA0101
transcript. (B) Transfection with #759si vector resulted in drastic reduction
of the numbers
of viable cells measured by the number of colony forination, compared with the
cells
transfected with siRNA expression vector in which did not showed their
knockdown effect on
KIAA0101. (C) Transfection with #759si vector resulted in drastic reduction of
the numbers
of viable cells measured by MTT assay. ABS on Y-axis at MTT assay means
absorbance at
490 nm, and at 630 nm as reference, measured with a microplate reader.
Fig. 11 The result of exogenous over-expression of KIAA0101 promoted cancer
cell growth and transformed NIH3T3. (A) Western blot analysis of six PK-45P
derivatives
cells (clones 1-6) expressing exogenous KIAA0101 constitutively and those
transfected with
mock vector (Mock 1-4). Exogenous introduction of KIAA0101 expression was
validated
with anti-HA tag antibody. ACTB served as a loading control. (B) The growth
measurement by MTT assay demonstrates that the six KIAA0101 clones (1-6, solid
lines)
grew significantly more rapidly than the two mock clones (1-4, dash lines). X-
, and Y-axis
represent day point after seeding and relative growth rate that was calculated
in absorbance of
the diameter by comparison with the absorbance value of day I as a control.
Each average is
plotted with error bars representing standard error. These experiences were in
triplicate
altogether. (C) Three KIAA0101-overexpressed clones and three mock clones were
established form NIH3T3 that did not express endogenous KIAA0101 mouse
homologue.
Three KIAA0101-overexpressed NIH3T3 clones (1-3) and mock NIH3T3 clones were
inoculated in the right and left flank of 8-week nude mice, respectively.
After four weeks,
only KIAA0101-overexpressed NIH3T3 cells formed the mass at the right frank of
nude mice.
(D) Each of the tumors was iminunostained by anti-KIAA0101 antibody, showing
the
exogenous KIAA0101 expression.
Fig. 12 KIAA0101 protein was associated with several DNA replication proteins.
(A) Immunoprecipitated fractions separated on SDS-PAGE gels followed by silver
staining
showed that several proteins were immunoprecipitated with KIAA0101 proteins
from cancer
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cell lysate, compared with results from a control sample. Each differential
band was
analyzed by a 1VIALDI-TOF system after in-gel trypsin digestion; they were
identified as
PCNA, POLD1 (polymerase S p125 subunit), and FEN1 (flap endonuclease-1). (B)
These
interactions were confirmed by immunoprecipitation experiment. All of these
proteins are
involved with DNA replication and POLD1 and FENl also bind to PCNA as well as
KIAA0101.
Fig. 13 The inhibition of the interaction between KIAA0101 and PCNA by cell-
permeable dominant-negative peptides. (A) Two dominant-negative peptides
(PIP20,
PIP16) containing PIP box ({q}KG{i}GE{ff}/SEQ ID NO: 21 shown in parentheses)
and its
mutant peptides (PIP20mt, PIP16mt) with the conserved PIP box residues
replaced with
alanines ({a}KG{a}GE{aa}/SEQ ID NO: 37 shown in parentheses) were designed and
conjugated them with arginine (R)-repeat to facilitate cell permeability. (B)
In vitro study,
immunoprecipitation validated the inhibition of the interaction between PCNA
and
KIAA0101 by PIP20 treatment, but PIP20mt and scramble peptides did not affect
the
interaction between PCNA and KIAA0101. (C) PIP20 treatment suppressed cell
growth of
KIAA0101 with KIAA0101 expression dose-dependently, while PIP20mut and
scramble
peptide did not. On the other hand, PIP20 did not affect the growth of mouse
normal cell
line NIH3T3 cells that did not express the homologue of human KIAA0101. (D)
ShortPIP
peptides (PIP16 and PIPl6mt) were designed by delete four residues of N- and C-
terminus
with PIP box motif maintained, and PIP 16 treatment suppressed cancer cell
growth strongly,
however, PIP 16 also affected NIH3T3 growth.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Small interfering RNA:
The present invention based in part on the surprising discovery that
inhibiting
expression of REG4 and/or KIAA0101 is effective in inhibiting the cellular
growth of various
cancer cells, including those involved in pancreatic cancer, prostate cancer,
breast cancer and
bladder cancer. In particular, it has been surprisingly discovered that PDAC
can be
prevented or inhibited by inhibiting REG4 gene. The inventions described in
this
application are based in part on these discoveries.
The invention provides methods for inhibiting cell growth. Among the methods
provided are those comprising contacting a cell with a composition comprising
a small
interfering RNA (siRNA) that inhibits expression (i.e., transcription or
translation) of REG4
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or KIAA0101. The invention also provides methods for inhibiting tumor cell
growth in a
subject. Such methods include administering to a subject a composition
comprising a small
interfering RNA (siRNA) that hybridizes specifically to a sequence from REG4
or KIAA0101.
Another aspect of the invention provides methods for inhibiting the expression
of the REG4
gene or KIAA0101 gene in a cell of a biological sample.
Expression of the gene may be inhibited by introduction of a double stranded
ribonucleic acid (RNA) molecule into the cell in an amount sufficient to
inhibit expression of
the REG4 gene or KIAA0101 gene. Another aspect of the invention relates to
products
including nucleic acid sequences and vectors as well as to compositions
comprising them,
useful, for example, in the provided methods. Ainong the products provided are
the siRNA
molecules having the property to inhibit expression of the REG4 gene or
KIAA0101 gene
when introduced into a cell expressing said gene. Among such molecules are
those that
comprise a sense strand and an antisense strand, wherein the sense strand
comprises a
ribonucleotide sequence corresponding to a REG4 or KIAA0101 target sequence,
and
wlierein the antisense strand comprises a ribonucleotide sequence which is
complementary to
said sense strand. The sense and the antisense strands of the molecule
hybridize to each
other to form a double-stranded molecule.
As used herein, the term "organism" refers to any living entity comprised of
at least
one cell. A living organism can be as simple as, for example, a single
eukaryotic cell or as
complex as a mammal, including a human being.
As used herein, the term "biological sample" refers to a whole organism or a
subset of
its tissues, cells or component parts (e.g. bodily fluids, including but not
limited to blood,
mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic
fluid, amniotic
cord blood, urine, vaginal fluid and semen). "Biological sample" further
refers to a
homogenate, lysate, extract, cell culture or tissue culture prepared from a
whole organism or a
subset of its cells, tissues or component parts, or a fraction or portion
thereof. Lastly,
"biological sample" refers to a medium, such as a nutrient broth or gel in
which an organism
has been propagated, which contains cellular coinponents, such as proteins or
nucleic acid
molecules.
The invention features methods of inhibiting cell growth. Cell growth can be
inhibited by contacting a cell with a composition of a small interfering RNA
(siRNA) of
REG4 or KIAA0101. The cell can be further contacted with a transfection-
enhancing agent.
The cell can be provided in vitro, in vivo or ex vivo. The subject can be a
mammal, e.g., a
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human, non-lluman primate, mouse, rat, dog, cat, horse, or cow. The cell can
be a pancreatic
ductal cell. Alternatively, the cell can be a tumor cell (i.e., cancer cell),
for example, a
carcinoma cell or an adenocarcinoma cell. For example, the cell can be a
pancreatic cancer
cell, especially pancreatic ductal adenocarcinoma cell, prostatic cancer cell,
breast cancer cell,
or a bladder cancer cell. By inhibiting cell growth is meant that the treated
cell proliferates
at a lower rate or has decreased viability than an untreated cell. Cell growth
is measured by
proliferation assays known in the art.
The term "polynucleotide" and "oligonucleotide" are used interchangeably
herein
unless otherwise specifically indicated and are referred to by their commonly
accepted single-
letter codes. The terms apply to nucleic acid (nucleotide) polymers in which
one or more
nucleic acids are linked by ester bonding. The polynucleotide or
oligonucleotide may be
composed of DNA, RNA or a combination thereof.
As use herein, the term "double-stranded molecule" refers to a nucleic acid
molecule
that inhibits expression of a target gene including, for example, short
interfering RNA
(siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA
(shRNA)) and
short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and
RNA
(dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
By the term "siRNA" is meant a double stranded RNA molecule which prevents
translation of a target mRNA. Standard tecliniques of introducing siRNA into
the cell are
used, including those in which DNA is a template from which RNA is
transcribed. The
siRNA includes a sense REG4 or KIAA0101 nucleic acid sequence, an antisense
REG4 or
KIAA0101 nucleic acid sequence or both. The siRNA may comprise two
complementary
molecules or may be constructed such that a single transcript has both the
sense and
complementary antisense sequences from the target gene, e.g., a hairpin,
which, in some
embodiments, leads to production of micro RNA (miRNA). The siRNA may either be
a
dsRNA or shRNA.
As used herein, the term "dsRNA" refers to a construct of two RNA molecules
comprising complementary sequences to one another and that have annealed
together via the
complementary sequences to form a double-stranded RNA molecule. The nucleotide
sequence of two strands may comprise not only the "sense" or "antisense" RNAs
selected
from a protein coding sequence of target gene sequence, but also RNA molecule
having a
nucleotide sequence selected from non-coding rigion of the target gene.
The term "shRNA", as used herein, refers to an siRNA having a stem-loop
structure,
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comprising a first and second regions complementary to one another, i.e.,
sense and antisense
strands. The degree of complementarity and orientation of the regions being
sufficient such
that base pairing occurs between the regions, the first and second regions
being joined by a
loop region, the loop resulting from a lack of base pairing between
nucleotides (or nucleotide
analogs) within the loop region. The loop region of an shRNA is a single-
stranded region
intervening between the sense and antisense strands and may also be referred
to as
"intervening single-strand".
As use herein, the term "siD/R-NA" refers to a double-stranded polynucleotide
molecule which is composed of both RNA and DNA, and includes hybrids and
chimeras of
RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid
indicates a
molecule wherein a polynucleotide composed of DNA and a polynucleotied
composed of
RNA hybridize to each other to form the double-stranded molecule; whereas a
chimera
indicates that one or both of the strands composing the double stranded
molecule may contain
RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are
used. The
siD/R-NA includes a CX sense nucleic acid sequence (also referred to as "sense
strand"), a
CX antisense nucleic acid sequence (also referred to as "antisense strand") or
both. The
siD/R-NA may be constructed such that a single transcript has both the sense
and
complementary antisense nucleic acid sequences from the target gene, e.g., a
hairpin. The
siD/R-NA may either be a dsD/R-NA or shD/R-NA.
As used herein, the term "dsD/R-NA" refers to a construct of two molecules
comprising complementary sequences to one another and that have annealed
together via the
complementary sequences to form a double-stranded polynucleotide molecule. The
nucleotide sequence of two strands may comprise not only the "sense" or
"antisense"
polynucleotides sequence selected from a protein coding sequence of target
gene sequence,
but also polynucleotide having a nucleotide sequnence selected from non-coding
region of the
target gene. One or both of the two molecules constructing the dsD/R-NA are
composed of
both RNA and DNA (chimeric molecule), or alternatively, one of the molecules
is composed
of RNA and the other is composed of DNA (hybrid double-strand).
The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop
structure, comprising a first and second regions complementary to one another,
i.e., sense and
antisense strands. The degree of complementarity and orientation of the
regions being
sufficient such that base pairing occurs between the regions, the first and
second regions being
joined by a loop region, the loop resulting from a lack of base pairing
between nucleotides (or
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nucleotide analogs) within the loop region. The loop region of an shD/R-NA is
a single-
stranded region intervening between the sense and antisense strands and may
also be referred
to as "intervening single-strand".
The method is used to alter gene expression in a cell in which expression of
REG4 or
KIAA0101 is aberrantly up-regulated, e.g., as a result of malignant
transformation of the cells.
Binding of the siRNA to a REG4 or KIAA0101 transcript in the target cell
results in a
reduction in REG4 or KIAA0101 production by the cell. The length of the
oligonucleotide
is at least about 10 nucleotides and may be as long as the naturally-occurring
REG4 or
KIAA0101 transcript. Preferably, the oligonucleotide is less than about 75,
about 50, or
about 25 nucleotides in length. Most preferably, the oligonucleotide is about
19 to about 25
nucleotides in length. Examples of the siRNA oligonucleotides of REG4 or
KIAA0101
which inhibit REG4 or KIAA0101 expression in mammalian cells include
oligonucleotides
containing target sequences, for example, nucleotide of SEQ ID NO: 5 or SEQ ID
NO: 32,
respectively.
Methods for designing double stranded RNA having the ability to inhibit gene
expression in a target cell are known. (See for example, US Patent No.
6,506,559, herein
incorporated by reference in its entirety). For example, a computer program
for designing
siRNAs is available from the Ambion website
(www.ambion.com/techlib/misc/siRNA-finder.html). The computer program
available from
Ambion, Inc. selects nucleotide sequences for siRNA synthesis based on the
following
protocol.
Selection of siRNA Tar eg t Sites
1. Beginning with the AUG start codon of the transcript, scan downstream for
AA
dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent
19
nucleotides as potential siRNA target sites. Tuschl et al., Genes Dev 13(24):
3191-7
(1999), don't recommend designing siRNA to the 5' and 3' untranslated regions
(UTRs) and regions near the start codon (within 75bases) as these may be
richer in
regulatory protein binding sites. UTR-binding proteins and/or translation
initiation
complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the appropriate genome database
(human, mouse,
rat, etc.) and eliminate from consideration any target sequences with
significant
homology to other coding sequences. It is suggested to use BLAST, which can be
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found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/
3. Select qualifying target sequences for synthesis. Selecting several target
sequences
along the length of the gene to evaluate is typical.
Also included in the invention are isolated nucleic acid molecules that
include the
nucleic acid sequence of target sequences, for example, nucleotide of SEQ ID
NO: 5 or SEQ
ID NO: 32, or a nucleic acid molecule that is complementary to the nucleic
acid sequence of
nucleotide of SEQ ID NO: 5 or SEQ ID NO: 32. As used herein, an "isolated
nucleic acid"
is a nucleic acid removed from its original environment (e.g., the natural
environment if
naturally occurring) and thus, synthetically altered from its natural state.
In the present
invention, isolated nucleic acid includes DNA, RNA, and derivatives thereof.
When the
isolated nucleic acid is RNA or derivatives thereof, base "t" should be
replaced with "u" in
the nucleotide sequences.
As used herein, the terin "complementary" refers to Watson-Crick or Hoogsteen
base pairing between nucleotides units of a nucleic acid molecule, and the
term "binding"
means the physical or chemical interaction between two nucleic acids or
compounds or
associated nucleic acids or compounds or combinations thereof. When the
polynucleotide
comprises modified nucleotides and/or non-phosphodiester linkages, these
polynucleotides
may also bind each other as same manner. Generally, complementary nucleic acid
sequences liybridize under appropriate conditions to form stable duplexes
containing few or
no mismatches. For the purposes of this invention, two sequences having 5 or
fewer
mismatches are considered to be complementary. Furthermore, the sense strand
and
antisense strand of the isolated nucleotide of the present invention, can form
double stranded
nucleotide or hairpin loop structure by the hybridization.
In a preferred embodiment, such duplexes contain no more than 1 mismatch for
every10 matches. In an especially preferred embodiment, where the strands of
the duplex
are fully complementary, such duplexes contain no mismatches. The nucleic acid
molecule
is less than 1518 nucleotides in length for REG4 or less than 1508 nucleotides
in length for
IUAA0101. For example, the nucleic acid molecule is less than about 500, about
200, or
about 75 nucleotides in length. Also included in the invention is a vector
containing one or
more of the nucleic acids described herein, and a cell containing the vectors.
The isolated
nucleic acids of the present invention are useful for siRNA against REG4 or
IUAA0101, or
DNA encoding the siRNA. When the nucleic acids are used for siRNA or coding
DNA
thereof, the sense strand is preferably longer than about 19 nucleotides, and
more preferably
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longer than 21 nucleotides.
The double-stranded molecules of the invention may contain one or more
modified
nucleotides and/or non-phosphodiester linkages. Chemical modifications well
known in the
art are capable of increasing stability, availability, and/or cell uptake of
the double-stranded
molecule. The skilled person will be aware of other types of chemical
modification which
may be incorporated into the present molecules (W003/070744; W02005/045037).
In one
embodiment, modifications can be used to provide improved resistance to
degradation or
improved uptake. Examples of such modifications include phosphorothioate
linkages, 2'-O-
methyl ribonucleotides (especially on the sense strand of a double-stranded
molecule), 2'-
deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base"
nucleotides, 5'-C-
methyl nucleotides, and inverted deoxyabasic residue incorporation
(US20060122137).
In another embodiment, modifications can be used to enhance the stability or
to
increase targeting efficiency of the double-stranded molecule. Modifications
include
chemical cross linking between the two complementary strands of a double-
stranded molecule,
chemical modification of a 3' or 5' terminus of a strand of a double-stranded
molecule, sugar
modifications, nucleobase modifications and/or backbone modifications, 2-
fluoro modified
ribonucleotides and 2'-deoxy ribonucleotides (W02004/029212). In another
embodiment,
modifications can be used to increased or decreased affinity for the
complementary
nucleotides in the target mRNA and/or in the complementary double-stranded
molecule strand
(W02005/044976). For example, an unmodified pyrimidine nucleotide can be
substituted
for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an
unmodified
purine can be substituted with a 7-deza, 7-alkyi, or 7-alkenyi purine. In
another embodiment,
when the double-stranded molecule is a double-stranded molecule with a 3'
overhang, the 3'-
terminal nucleotide overhanging nucleotides may be replaced by
deoxyribonucleotides
(Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further
details, published
documents such as US20060234970 are available. The present invention is not
limited to
these examples and any known chemical modifications may be employed for the
double-
stranded molecules of the present invention so long as the resulting molecule
retains the
ability to inhibit the expression of the target gene.
Furthermore, the double-stranded molecules of the invention may comprise both
DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide
of a
DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows
increased
stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule
consisting
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of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera
type
double-stranded molecule comprising both DNA and RNA on any or both of the
single
strands (polynucleotides), or the like may be formed for enhancing stability
of the double-
stranded molecule. The hybrid of a DNA strand and an RNA strand may be the
hybrid in
which either the sense strand is DNA and the antisense strand is RNA, or the
opposite so long
as it has an activity to inhibit expression of the target gene when introduced
into a cell
expressing the gene. Preferably, the sense strand polynucleotide is DNA and
the antisense
strand polynucleotide is RNA. Also, the chimera type double-stranded molecule
may be
either where both of the sense and antisense strands are composed of DNA and
RNA, or
where any one of the sense and antisense strands is composed of DNA and RNA so
long as it
has an activity to inhibit expression of the target gene when introduced into
a cell expressing
the gene.
In order to enhance stability of the double-stranded molecule, the molecule
preferably
contains as much DNA as possible, whereas to induce inhibition of the target
gene expression,
the nlolecule is required to be RNA within a range to induce sufficient
inhibition of the
expression. As a preferred example of the chimera type double-stranded
molecule, an
upstream partial region (i.e., a region flanking to the target sequence or
complementary
sequence thereof within the sense or antisense strands) of the double-stranded
molecule is
RNA. Preferably, the upstream partial region indicates the 5' side (5'-end) of
the sense
strand and the 3' side (3'-end) of the antisense strand. That is, in
preferable embodiments, a
region flanking to the 3'-end of the antisense strand, or both of a region
flanking to the 5'-end
of sense strand and a region flanking to the 3'-end of antisense strand
consists of RNA. For
instance, the chimera or hybrid type double-stranded molecule of the present
invention
comprise following combinations.
sense strand: 5'-[DNA]-3'
3'-(RNA)-[DNA]-5' : antisense strand,
sense strand: 5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5': antisense strand, and
sense strand: 5'-(RNA)-[DNA]-3'
3'-(RNA)-5' : antisense strand.
The upstream partial region preferably is a domain consisting of 9 to 13
nucleotides
counted from the terminus of the target sequence or complementary sequence
thereto within
the sense or antisense strands of the double-stranded molecules. Moreover,
preferred
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examples of such chimera type double-stranded molecules include those having a
strand
length of 19 to 21 nucleotides in which at least the upstream half region (5'
side region for the
sense strand and 3' side region for the antisense strand) of the
polynucleotide is RNA and the
other half is DNA. In such a chimera type double-stranded molecule, the effect
to inhibit
expression of the target gene is much higher when the entire antisense strand
is RNA
(US20050004064).
In the present invention, the double-stranded molecule may form a hairpin,
such as a
short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-
NA).
The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a
tight
hairpin turn that can be used to silence gene expression via RNA interference.
The shRNA
or shD/R-NA comprises the sense target sequence and the antisense target
sequence on a
single strand wherein the sequences are separated by a loop sequence.
Generally, the hairpin
structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which
is then bound
to the RNA-induced silencing complex (RISC). This complex binds to and cleaves
mRNAs
which match the target sequence of the dsRNA or dsD/R-NA.
The invention is based in part on the discovery that the gene encoding REG4 is
over-
expressed in pancreatic ductal adenocarcinoma (PDACa) compared to non-
cancerous
pancreatic tissue, and that the gene encoding KIA0101 is over-expressed in
pancreatic cancer,
prostatic cancer cell, breast cancer cell, and bladder cancer cell compared to
non-cancerous
each tissue. The cDNA of REG4 is 1518 nucleotides in length. On the other
hand, the
cDNA of KIAA0101 is 1508 nucleotides in length. The nucleic acid and
polypeptide
sequences of REG4 (Genbank accession No: AY126670) are shown in SEQ ID NO: 1
and 2,
respectively, and that of KIAA0101 (Genbank accession No: NM 014736) are shown
in SEQ
ID NO: 39 and 40, respectively.
The present invention discloses that transfection of siRNA comprising SEQ ID
NO: 5
resulted in growth inhibition of PDAC cell lines. Furthermore, transfection of
siRNA
comprising SEQ ID NO: 32 resulted in a growth inhibition of pancreatic cancer
cell lines.
Methods of inhibiting cell growth
The present invention relates to inhibiting cell growth, i.e., cancer cell
growth by
inhibiting expression of REG4 or IfIAA0101. Expression of REG4 or IUAA0101 is
inhibited, for example, by small interfering RNA (siRNA) that specifically
target the REG4
gene or KIAA0101 gene. REG4 targets include, for example, nucleotide of SEQ ID
NO: 5,
and KIAA0101 targets similarly include nucleotide of SEQ ID NO: 32.
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The term "specifically inhibit" in the context of inhibitory polynucleotides
and
polypeptides refers to the ability of an agent or ligand to inhibit the
expression or the
biological function of REG4 and/or KIAA0101. Specific inhibition typically
results in at
least about a 2-fold inhibition over background, preferably greater than about
10-fold and
most preferably greater than 100-fold inhibition of REG4 and/or KIAA0101
expression (e.g.,
transcription or translation) or measured biological function (e.g., cell
growth or proliferation,
inhibition of apoptosis, intracellular signaling from REG4, for example,
activation of the EGF
receptor/Akt/AP-1 signaling pathway with respect to REG4; binding to PCNA or
another
intracellular protein with respect to KIAA0101). Expression levels and/or
biological
function can be measured in the context of comparing treated and untreated
cells, or a cell
population before and after treatment. In some embodiments, the expression or
biological
function of REG4 and/or KIAA0101 is completely inhibited. Typically, specific
inhibition
is a statistically meaningful reduction in REG4/KIAA0101 expression or
biological function
(e.g., p< 0.05) using an appropriate statistical test.
In non-mammalian cells, double-stranded RNA (dsRNA) has been shown to exert a
strong and specific silencing effect on gene expression, which is referred as
RNA interference
(RNAi) (Sharp PA. Genes Dev. 1999 Jan 15;13(2):139-41.). dsRNA is processed
into 20-23
nucleotides dsRNA called small interfering RNA (siRNA) by an enzyme containing
RNase
III motif. The siRNA specifically targets complementary mRNA with a
multicomponent
nuclease complex (Hammond SM, et al. Nature. 2000 Mar 16; 404 (6775): 293- 6;
Hannon
GJ. Nature. 2002 Jul 11; 418 (6894): 244-51.). In mammalian cells, siRNA
composed of 20
or 21-mer dsRNA with 19 complementary nucleotides and 3' terminal
noncomplementary
dimmers of thymidine or uridine, have been shown to have a gene specific knock-
down effect
without inducing global changes in gene expression (Elbashir SM, et al.
Nature. 2001 May
24;411(6836):494-8.).
The growth of cells is inhibited by contacting a cell, with a composition
containing
a siRNA of REG4 or KIAA0101. The cell is further contacted with a transfection
agent.
Suitable transfection agents are known in the art. By inhibition of cell
growth is meant the
cell proliferates at a lower rate or has decreased viability compared to a
cell not exposed to the
composition. Cell growth is measured by methods known in the art such as, the
MTT cell
proliferation assay.
The siRNA of REG4 or KIAA0101 is directed to a single target of REG4 gene
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sequence or KIAA0101 gene sequence, respectively. Alternatively, the siRNA is
directed to
multiple targets of REG4 or KIAA0101 gene sequences. For example, the
composition
contains siRNA of REG4 or KIAA0101 directed to two, three, four, or five or
more target
sequences of REG4 or KIAA0101, respectively. By REG4 or KIAA0101 target
sequence is
meant a nucleotide sequence that is identical to a portion of the REG4 gene or
the KIAA0101
gene (i. e, a polynucleotide within a REG4 or KIAA0101 gene that is equal in
length to and
complementary to an siRNA). The target sequence can include the 5'
untranslated (UT)
region, the open reading frame (ORF) or the 3' untranslated region of the
lluman REG4 or
KIAA0101 gene. Alternatively, the siRNA is a nucleic acid sequence
complementaryto an
upstream or downstream modulator of REG4 or KIAA0101 gene expression. Examples
of
upstream and downstream modulators include, a transcription factor that binds
the REG4 or
KIAA0101 gene promoter, a kinase or phosphatase that interacts with the REG4
or
KIAA0101 polypeptide, a REG4 or KIAA0101 promoter or enhancer.
The siRNA of REG4 or KIAA0101 which hybridize to target mR.NA decreases or
inhibits production of the REG4 or KIAA0101 polypeptide product encoded by the
REG4 or
KIAA0101 gene by associating with the normally single-stranded mRNA
transcript, thereby
interfering with translation and thus, suppresses the expression of the
protein. Thus, the
siRNA molecules of the invention can be defined by their ability to hybridize
specifically to
mRNA or cDNA from a REG4 or KIAA0101 gene under stringent conditions.
For the purposes of this invention the terms "hybridize" or "hybridize
specifically"
are used to refer the ability of two nucleic acid molecules to hybridize under
"stringent
hybridization conditions." The phrase "stringent hybridization conditions"
refers to
conditions under which a nucleic acid molecule will hybridize to its target
sequence, typically
in a complex mixture of nucleic acids, but not detectably to other sequences.
Stringent
conditions are sequence-dependent and will be different in different
circumstances. Longer
sequences hybridize specifically at higher temperatures. An extensive guide to
the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biocherraishy afzdMolecular
Biology--Hybridization with Nucleic Probes, "Overview of principles of
liybridization and the
strategy of nucleic acid assays" (1993). Generally, stringent conditions are
selected to be
about 5-10 C lower than the thermal melting point (T,,,) for the specific
sequence at a defined
ionic strength pH. The T,,, is the temperature (under defined ionic strength,
pH, and nucleic
concentration) at which 50% of the probes complementary to the target
hybridize to the target
sequence at equilibrium (as the target sequences are present in excess, at
T,,,, 50% of the
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probes are occupied at equilibrium). Stringent conditions may also be achieved
with the
addition of destabilizing agents such as formamide. For selective or specific
hybridization, a
positive signal is at least two times background, preferably 10 times
background hybridization.
Exemplary stringent hybridization conditions can be as following: 50%
formamide, 5x SSC,
and 1% SDS, incubating at 42 C, or, 5x SSC, 1% SDS, incubating at 65 C, with
wash in 0.2x
SSC, and 0.1% SDS at 50 C.
The siRNA of the invention is less than about 500, about 200, about 100, about
50,
or about 25 nucleotides in length. Preferably the siRNA is about 19 to about
25 nucleotides
in length. Exemplary nucleic acid sequence for the production of REG4 siRNA
includes the
sequence of nucleotide of SEQ ID NO: 5 as the target sequence. Similarly,
nucleic acid
sequence for the production of KIAA0101 siRNA include the sequence of
nucleotide of SEQ
ID NO:32 as the target sequence. Furthermore, in order to enhance the
inhibition activity of
the siRNA, nucleotide "u" can be added to 3'end of the antisense strand of the
target sequence.
The number of "u"s to be added is at least about 2, generally about 2 to about
10, preferably
about 2 to about 5. The added "u"s form single strand at the 3'end of the
antisense strand of
the siRNA.
The cell is any cell that expresses or over-expresses REG4 or KIAA0101. The
cell is
an epithelial cell such as a pancreatic ductal cell. Alternatively, the cell
is a tumor cell such
as a carcinoma, adenocarcinoma, blastoma, leukemia, myeloma, or sarcoma. The
cell is a
pancreatic cancer cell, especially pancreatic ductal adenocarcinoma cell,
prostatic cancer cell,
breast cancer cell, and bladder cancer cell.
The siRNA of REG4 or KIA.A0101 is directly introduced into the cells in a form
that
is capable of binding to the mRNA transcripts. Alternatively, the DNA encoding
the siRNA
of REG4 or IUAA0101 is in a vector.
Vectors are produced for example by cloning a REG4 or KIAA0101 target sequence
into an expression vector operatively-linked regulatory sequences flanking the
REG4 or
ICAA0101 sequence in a manner that allows for expression (by transcription of
the DNA
molecule) of both strands (Lee, N.S., et al. Nature Biotechnology 20 : 500-
5.). An RNA
molecule that is antisense to REG4 or KIAA0101 mRNA is transcribed by a first
promoter
(e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is
the sense
strand for the REG4 or KIAA0101 mRNA is transcribed by a second promoter
(e.g., a
promoter sequence 5' of the cloned DNA). The sense and antisense strands
hybridize in vivo
to generate the siRNA constructs for silencing of the REG4 or KIAA0101 gene.
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Alternatively, two constructs are utilized to create the sense and anti-sense
strands of a siRNA
construct. Cloned REG4 or KIAA0101 can encode a construct having secondary
structure,
e.g., hairpins, wherein a single transcript has both the sense and
complementary antisense
sequences from the target gene.
A loop sequence consisting of an arbitrary nucleotide sequence can be located
between the sense and antisense sequence in order to form the hairpin loop
structure. Thus,
the present invention also provides the siRNA having the general formula 5'-
[A]-[B]-[A']-3',
wherein [A] is a ribonucleotide sequence corresponding to a sequence that
specfically
hybridizes to an mRNA or a cDNA from REG4 or KIAA0101. In preferred
embodiments,
[A] is a ribonucleotide sequence corresponding to a sequence of nucleotides of
SEQ ID NO: 5
or SEQ ID NO:32.
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and
[A] is a ribonucleotide sequence consisting of the complementary sequence of
[A]
The region [A] hybridizes to [A'], and then a loop consisting of region [B] is
formed.
The loop sequence may be preferably about 3 to about 23 nucleotide in length.
The loop
sequence, for example, can be selected from group consisting of following
sequences
(www.ainbion.com/techlib/tb/tb_506.html). Furthermore, loop sequence
consisting of 23
nucleotides also provides active siRNA (Jacque, J.M., et al. (2002) Nature 418
: 435-438.).
CCC, CCACC or CCACACC: Jacque, J.M., et al. (2002) Nature, Vol. 418: 435-8.
UUCG: Lee, N.S., et al. (2002) Nature Biotechnology 20 : 500-5. Fruscoloni,
P., et
al. (2003) Proc. Natl. Acad. Sci. USA 100(4): 1639-44.
UUCAAGAGA: Dykxhoorn, D. M., et al. Nature Reviews Molecular Cell Biology 4:
457-67.
For exainple, preferable siRNAs having hairpin loop structure of the present
invention
are showm below. In the following structure, the loop sequence can be selected
from group
consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Preferable loop
sequence is UUCAAGAGA ("ttcaagaga" in DNA).
5'-GACAGAAGGAAGAAACTCA-[B]- TGAGTTTCTTCCTTCTGTC-3' (for target
sequence of SEQ ID NO:5)
The regulatory sequences flanking the REG4 or KIAA0101 sequence are identical
or
are different, such that their expression can be modulated independently, or
in a temporal or
spatial manner. siRNAs are transcribed intracellularly by cloning the REG4 or
KIAA0101
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gene templates into a vector containing, e.g., a RNA polymerase III
transcription unit from
the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter. For introducing
the
vector into the cell, transfection-enhancing agent can be used. FuGENE (Roche
Diagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen),
and
Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing
agent.
Oligonucleotides and oligonucleotides complementary to various portions of
REG4 or
KIAA0101 mRNA were tested in viti-o for their ability to decrease production
of REG4 or
KIAA0101 in tumor cells (e.g., using the pancreatic cell line such as
pancreatic cancer cell
line or pancreatic ductal adenocarcinoma (PDAC) cell line) according to
standard methods.
A reduction in REG4 or KIAA0101 gene product in cells contacted with the
candidate siRNA
composition compared to cells cultured in the absence of the candidate
composition is
detected using specific antibodies of REG4 or KIAA0101, or other detection
strategies.
Sequences which decrease production of REG4 or KIAA0101 in in vitro cell-based
or cell-
free assays are then tested for there inhibitory effects on cell growth.
Sequences which
inhibit cell growth in vitro cell-based assay are test in vivo in rats or mice
to confirm
decreased REG4 or KIAA0101 production and decreased tumor cell growth in
animals with
malignant neoplasms.
Methods of treating malignant tumors
Patients with tumors characterized as over-expressing REG4 or KIAA0101 are
treated by administering siRNA of REG4 or KIAA0101, respectively. siRNA
therapy is
used to inhibit expression of REG4 or KIAA0101 in patients suffering from or
at risk of
developing, for exainple, pancreatic cancer, especially pancreatic ductal
adenocarcinoma
(PDAC), prostatic cancer cell, breast cancer cell, and bladder cancer cell.
Such patients are
identified by standard methods of the particular tumor type. Pancreatic
cancer, pancreatic
ductal adenocarcinoma (PDAC), prostatic cancer cell, breast cancer cell, and
bladder cancer
cell is diagnosed for example, by CT, MRI, ERCP, MRCP, computer tomography, or
ultrasound. Treatment is efficacious if the treatment leads to clinical
benefit such as, a
reduction in expression of REG4 or KIAA0101, or a decrease in size,
prevalence, or
metastatic potential of the tumor in the subject. When treatment is applied
prophylactically,
"efficacious" means that the treatment retards or prevents tumors from forming
or prevents or
alleviates a clinical symptom of the tumor. Efficaciousness is determined in
association with
any known method for diagnosing or treating the particular tumor type.
siRNA therapy is carried out by administering to a patieiit an siRNA by
standard
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vectors encoding the siRNAs of the invention and/or gene delivery systems such
as by
delivering the synthetic siRNA molecules. Typically, synthetic siRNA molecules
are
chemically stabilized to prevent nuclease degradation in vivo. Methods for
preparing
chemically stabilized RNA molecules are well known in the art. Typically, such
molecules
comprise modified backbones and nucleotides to prevent the action of
ribonucleases. Other
modifications are also possible, for example, cholesterol-conjugated siRNAs
have shown
improved pharmacological properties. (Song et al. Nature Med. 9:347-351
(2003)).
Suitable gene delivery systems may include liposomes, receptor-mediated
delivery
systems, or viral vectors such as herpes viruses, retroviruses, adenoviruses
and adeno-
associated viruses, among others. A therapeutic nucleic acid composition is
forinulated in a
pharmaceutically acceptable carrier. The therapeutic composition may also
include a gene
delivery system as described above. Pharmaceutically acceptable carriers are
biologically
compatible vehicles which are suitable for administration to an animal, e.g.,
physiological
saline. A therapeutically effective amount of a compound is an amount which is
capable of
producing a medically desirable result such as reduced production of a REG4 or
KIAA0101
gene product, reduction of cell growth, e.g., proliferation, or a reduction in
tumor growth in a
treated animal.
Parenteral administration, such as intravenous, subcutaneous, intramuscular,
and
intraperitoneal delivery routes, may be used to deliver siRNA compositions of
REG4 or
IKIAA0101. For treatment of pancreatic, prostatic, breast, and bladder tumors,
direct
infusion into the tissue or near the site of cancer, is useful.
Dosages for any one patient depends upon many factors, including the patient's
size,
body surface area, age, the particular nucleic acid to be administered, sex,
time and route of
administration, general health, and other drugs being administered
concurrently. Dosage for
intravenous administration of nucleic acids is from approximately 106 to 1022
copies of the
nucleic acid molecule.
The polynucleotides are administered by standard methods, such as by injection
into
the interstitial space of tissues such as inuscles or skin, introduction into
the circulation or into
body cavities or by inhalation or insufflation. Polynucleotides are injected
or otherwise
delivered to the animal with a pharmaceutically acceptable liquid carrier,
e.g., a liquid carrier,
which is aqueous or partly aqueous. The polynucleotides are associated with a
liposome
(e.g., a cationic or anionic liposome). The polynucleotide includes genetic
information
necessary for expression by a target cell, such as a promoter.
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Anti-REG4 Antibodies and Antigen Binding Proteins:
Antibodies
The present inventors have shown that a monoclonal antibody against REG4
neutralized its growth-promoting effects of REG4 and attenuated significantly
the growth of
PDAC cells. The results revealed that treatment of disease associated with
REG4-expressing
cells, for example, pancreatic cancer is conveniently carried out using
antibodies that bind to
REG4.
The present invention relates to pharmaceutical compositions for treating or
preventing cancers mediated by aberrant over-expression of REG4, including
pancreatic
cancer, said composition comprising a pharmaceutically effective amount of an
antibody or
fragment thereof, or an antigen binding protein, that binds to a protein
encoded by REG4 as
an active ingredient, and a pharmaceutically acceptable carrier. The present
invention also
relates to use of an anti-REG4 antibody or antigen binding protein to produce
pharmaceutical
compositions for treating or preventing pancreatic cancer. The pharmaceutical
compositions
of the present invention comprise anti-REG4 antibodies or antigen binding
proteins and
pharmaceutically acceptable carriers.
An "isolated" or "purified" polypeptide is a polypeptide that is substantially
free of
cellular material such as carbohydrate, lipid, or other contaminating proteins
from the cell or
tissue source from which the protein is derived, or substantially free of
chemical precursors or
other chemicals when chemically synthesized. The term "substantially free of
cellular
material" includes preparations of a polypeptide in which the polypeptide is
separated from
cellular components of the cells from which it is isolated or recombinantly
produced.
Thus, a polypeptide that is substantially free of cellular material includes
preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by
dry weight) of
heterologous protein (also referred to herein as a "contaminating protein").
When the
polypeptide is recombinantly produced, it is also preferably substantially
free of culture
medium, which includes preparations of polypeptide with culture medium less
than about
20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide
is
produced by chemical synthesis, it is preferably substantially free of
chemical precursors or
other chemicals, which includes preparations of polypeptide with chemical
precursors or other
chemicals involved in the synthesis of the protein less than about 30%, 20%,
10%, 5% (by dry
weiglit) of the volume of the protein preparation. That a particular protein
preparation
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contains an isolated or purified polypeptide can be shown, for example, by the
appearance of
a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of
the protein preparation and Coomassie Brilliant Blue staining of the gel. In a
preferred
embodiment, antibodies of the present invention or fragments thereof are
isolated or purified.
An "isolated" or "purified" nucleic acid molecule is one which is separated
from
other nucleic acid molecules which are present in the natural source of the
nucleic acid
molecule. An "isolated" or "purified" nucleic acid molecule, such as a cDNA
molecule, can
be substantially free of other cellular material, or culture medium when
produced by
recombinant techniques, or substantially free of chemical precursors or other
chemicals when
chemically synthesized. In a preferred embodiment, nucleic acid molecules
encoding
antibodies of the present invention or fragments thereof are isolated or
purified.
"Antibodies" and "immunoglobulins" are glycoproteins having the same
structural
characteristics. While antibodies exhibit binding specificity to a specific
antigen,
immunoglobulins include both antibodies and other antibody-like molecules, for
which
antigen specificity has not been defined. Polypeptides of the latter kind are,
for example,
produced at low levels by the lymph system and at increased levels by
myelomas.
"Native antibodies and immunoglobulins" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical light (L)
chains and two
identical heavy (H) chains. Each light chain is linked to a heavy chain by one
covalent
disulfide bond, while the number of disulfide linkages varies between the
heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a variable
domain (VH)
followed by a number of constant domains (CH). Each light chain has a variable
domain at
one end (VL) and a constant domain at its other end (CL); the constant domain
of the light
chain is aligned with the first constant domain of the heavy chain, and the
light chain variable
domain is aligned with the variable domain of the heavy chain. Particular
amino acid
residues are believed to form an interface between the light- and heavy-chain
variable
domains (Chothia et al., (1985) J Mol Biol.;186;651-63; Novotny and Haber,
(1985) Proc
Natl Acad Sci USA.;82:4592-6).
The term "variable" refers to the fact that certain portions of the variable
domains
differ extensively in sequence among antibodies and are used in the binding
and specificity of
each particular antibody for its particular antigen. However, the variability
is not evenly
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distributed tliroughout the variable domains of antibodies. It is concentrated
in three
segments called complementarity-determining regions (CDRs) or hypervariable
regions both
in the light-chain and the heavy-chain variable domains. The more highly
conserved
portions of variable domains are called the framework (FR). The variable
domains of native
heavy and light chains each comprise four framework regions, largely adopting
a(3-sheet
configuration, connected by three CDRs, which form loops connecting, and in
some cases
forming part of the (3-sheet structure. The CDRs in each chain are held
together in close
proximity by the framework regions and, with the CDRs from the other chain,
contribute to
the formation of the antigen-binding site of antibodies (Kabat et al., (1991)
Sequences of
Proteins of Immunological Interest, Fifth Edition, National Institute of
Health, Bethesda, Md.).
The constant domains are not involved directly in binding an antibody to an
antigen but
exhibit various effector functions, such as participation of the antibody in
antibody-dependent
cellular toxicity.
Papain digestion of antibodies produces two identical antigen-binding
fragments,
called "Fab" fragments, each with a single antigen-binding site, and a
residual "Fc" fragment.
Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding
sites. "Fv" is the
minimum antibody fragment which contains a complete antigen-recognition and -
binding site.
This region consists of a dimer of one heavy- and one light-chain variable
domain in tight,
non-covalent association. It is in this configuration that the three CDRs of
each variable
domain interact to define an antigen-binding site on the surface of the VH-VL
dimer.
Collectively, the six CDRs confer antigen-binding specificity to the antibody.
However,
even a single variable domain (or half of an Fv comprising only three CDRs
specific for an
antigen) has the ability to recognize and bind antigen, although at a lower
affinity than the
entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first
constant domain (CH-1) of the heavy chain. Fab' fragments differ from Fab
fragments by
the addition of a few residues at the carboxy terminus of the heavy chain CH-1
domain
including one or more cysteines from the antibody hinge region. Fab'-SH is the
designation
herein for Fab', in which the cysteine residue(s) of the constant domains bear
a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs of Fab'
fragments
which have hinge cysteines between them. Other chemical couplings of antibody
fragments
are also kiiown.
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The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can
be assigned to one of two clearly distinct types, called x(kappa) and k
(lambda), based on the
amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins can be assigned to different classes. There are five
major classes
of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be
further
divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and
IgA2. The
heavy-chain constant domains that correspond to the different classes of
immunoglobulins
are called a, S, c, -y, and , respectively. The subunit structures and tlu-ee-
dimensional
configurations of different classes of immunoglobulins are well known.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from
a population of substantially homogeneous antibodies, i.e., the individual
antibodies
comprising the population are identical except for possible naturally
occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly specific,
being directed
against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal)
antibody preparations, which typically include different antibodies directed
against different
determinants (epitopes), each monoclonal antibody is directed against a single
determinant on
the antigen. In addition to their specificity, the monoclonal antibodies are
advantageous in
that they can be synthesized by hybridoma culture, uncontaminated by other
immunoglobulins. Tlius, the modifier "monoclonal" indicates the character of
the antibody
as being obtained from a substantially homogeneous population of antibodies,
and is not to be
construed as requiring production of the antibody by any particular method.
For example,
the monoclonal antibodies to be used in accordance with the present invention
can be made by
the hybridoma method first described by Kohler and Milstein, (1975)
Nature.;256:495-7, or
can be made by recombinant DNA methods (Cabilly et al., (1984) Proc Natl Acad
Sci
USA.;81:3273-7).
The monoclonal antibodies herein specifically include "chimeric" antibodies or
immunoglobulins, in which a portion of the heavy and/or liglit chain is
identical with or
homologous to corresponding sequences in antibodies derived from a particular
species or
belonging to a particular antibody class or subclass, while the remainder of
the chain(s) is
identical with or homologous to corresponding sequences in antibodies derived
from another
species or belonging to another antibody class or subclass, as well as
fragments of such
antibodies, so long as they exhibit the desired biological activity (Cabilly
et al., supra;
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Morrison et al., (1984) Proc Natl Acad Sci USA.;81:6851-5). Most typically,
chimeric
antibodies or immunoglobulins comprise human and murine antibody fragments,
generally
human constant and mouse variable regions.
"Humanized" forms of non-human (e.g., murine) antibodies are specific chimeric
immunoglobulins, immunoglobulin chains or fragments thereof which contain
minimal
sequence derived from non-human immunoglobulin. Such fragments also includes
Fv, Fab,
Fab', F(ab')2, or other antigen-binding subsequences of antibodies. For the
most part,
humanized antibodies are human immunoglobulins (recipient antibody) in which
residues
from a complementarity-determining region (CDR) of the recipient are replaced
by residues
derived from a CDR of a non-human species (donor antibody) such as mouse, rat,
or rabbit
having the desired specificity, affinity, and capacity.
In some instances, Fv framework residues of the human immunoglobulin may be
replaced by corresponding non-human residues. In the present invention, few,
two, or
preferably one of framework(s) in the humanized antibody may be replaced by
that of non-
human residues. Furthermore, humanized antibodies can comprise residues which
are found
neither in the recipient antibody nor in the imported CDR or framework
sequences. These
modifications are made to further refine and optimize antibody performance. In
general, the
humanized antibody will comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-
human immunoglobulin and all or substantially all of the framework regions are
those of a
human immunoglobulin consensus sequence.
The humanized antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
For
further details see Jones et al., (1986) Nature.;321:522-5; Riechmann et al.,
(1988)
Nature.;332:323-7; Presta, (1992) Curr Opin Struct Biol. 2:593-6.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein these domains are present in a single polypeptide chain.
Preferably, the
Fv polypeptide further comprises a polypeptide linker between the VH and VL
domains
which enables the sFv to form the desired structure for antigen binding. A
number of
methods have been described to discern chemical structures for converting the
naturally
aggregated but chemically separated light and heavy polypeptide chains from an
antibody V
region into an sFv molecule which will fold into a three dimensional structure
substantially
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similar to the structure of an antigen-binding site (U.S. Pat. Nos. 5,091,513,
5,132,405, and
4,946,778; Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenberg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).
Antigen Binding Proteins
The present invention invention also includes antigen binding proteins or non-
antibody binding proteins (e.g., ligands) that specifically bind to REG4 or
KIAA0101. Non-
antibody ligands include antibody mimics that use non-immunoglobulin protein
scaffolds,
including adnectins, avimers, single chain polypeptide binding molecules, and
antibody-like
binding peptidomiinetics, as discussed in more detail below.
Other compounds have been developed that target and bind to targets in a
manner
similar to antibodies. Certain of these "antibody mimics" use non-
immunoglobulin protein
scaffolds as alternative protein frameworks for the variable regions of
antibodies.
For example, Ladner et al. (U.S. Patent No. 5,260,203) describe single
polypeptide
chain binding molecules with binding specificity similar to that of the
aggregated, but
molecularly separate, light and heavy chain variable region of antibodies. The
single-chain
binding molecule contains the antigen binding sites of both the heavy and
light variable
regions of an antibody connected by a peptide linker and will fold into a
structure similar to
that of the two peptide antibody. The single-chain binding molecule displays
several
advantages over conventional antibodies, including, smaller size, greater
stability and are
more easily modified.
Ku et al. (Proc. Natl. Acad. Sci. U.S.A. 92(14):6552-6556 (1995)) discloses an
alternative to antibodies based on cytochrome b562. Ku et al. (1995) generated
a library in
which two of the loops of cytochrome b562 were randomized and selected for
binding against
bovine serum albumin. The individual mutants were found to bind selectively
with BSA
similarly with anti-BSA antibodies.
Lipovsek et al. (U.S. Patent Nos. 6,818,418 and 7,115,396) discloses an
antibody
mimic featuring a fibronectin or fibronectin-like protein scaffold and at
least one variable loop.
Known as Adnectins, these fibronectin-based antibody mimics exhibit many of
the same
characteristics of natural or engineered antibodies, including high affinity
and specificity for
any targeted ligand. Any technique for evolving new or improved binding
proteins can be
used with these antibody mimics.
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The structure of these fibronectin-based antibody mimics is similar to the
structure
of the variable region of the IgG heavy chain. Therefore, these mimics display
antigen
binding properties similar in nature and affinity to those of native
antibodies. Further, these
fibronectin-based antibody mimics exhibit certain benefits over antibodies and
antibody
fragments. For example, these antibody mimics do not rely on disulfide bonds
for native
fold stability, and are, therefore, stable under conditions which would
normally break down
antibodies. In addition, since the structure of these fibronectin-based
antibody mimics is
similar to that of the IgG heavy chain, the process for loop randomization and
shuffling can be
employed in vitro that is similar to the process of affinity maturation of
antibodies in vivo.
Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999)) discloses
an
antibody mimic based on a lipocalin scaffold (Anticalin ). Lipocalins are
composed of a(3-
barrel with four hypervariable loops at the terminus of the protein. Beste
(1999), subjected
the loops to random mutagenesis and selected for binding with, for example,
fluorescein.
Three variants exhibited specific binding with fluorescein, with one variant
showing binding
similar to that of an anti-fluorescein antibody. Further analysis revealed
that all of the
randomized positions are variable, indicating that Anticalin would be
suitable to be used as
an alternative to antibodies.
Anticalins are small, single chain peptides, typically between 160 and 180
residues, which provides several advantages over antibodies, including
decreased cost of
production, increased stability in storage and decreased immunological
reaction.
Hamilton et al. (U.S. Patent No. 5,770,380) discloses a synthetic antibody
mimic
using the rigid, non-peptide organic scaffold of calixarene, attached with
multiple variable
peptide loops used as binding sites. The peptide loops all project from the
same side
geometrically from the calixarene, with respect to each other. Because of this
geometric
confirmation, all of the loops are available for binding, increasing the
binding affinity to a
ligand. However, in comparison to other antibody mimics, the calixarene-based
antibody
mimic does not consist exclusively of a peptide, and therefore it is less
vulnerable to attack by
protease enzymes. Neither does the scaffold consist purely of a peptide, DNA
or RNA,
meaning this antibody mimic is relatively stable in extreme environmental
conditions and has
a long life span. Further, since the calixarene-based antibody mimic is
relatively small, it is
less likely to produce an immunogenic response.
Murali et al. (Cell. Mol. Biol. 49(2):209-216 (2003)) discusses a methodology
for
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reducing antibodies into smaller peptidomimetics, they term "antibody like
binding
peptidomemetics" (ABiP) which can also be useful as an alternative to
antibodies.
Silverman et al. (Nat. Biotechnol. (2005), 23: 1556-1561) discloses fusion
proteins
that are single-chain polypeptides comprising multiple domains termed
"avimers."
Developed from human extracellular receptor domains by in vitro exon shuffling
and phage
display the avimers are a class of binding proteins somewhat similar to
antibodies in their
affinities and specificities for various target molecules. The resulting
multidomain proteins
can comprise multiple independent binding domains that can exhibit improved
affinity (in
some cases sub-nanomolar) and specificity compared with single-epitope binding
proteins.
Additional details concerning methods of construction and use of avimers are
disclosed, for
example, in U.S. Patent App. Pub. Nos. 20040175756, 20050048512, 20050053973,
20050089932 and 20050221384.
In addition to non-immunoglobulin protein frameworks, antibody properties have
also been mimicked in compounds comprising RNA molecules and unnatural
oligoiners (e.g.,
protease inhibitors, benzodiazepines, purine derivatives and beta-turn mimics)
all of which are
suitable for use with the present invention.
Neutralizing activity
Antibodies and non-antibody binding proteins exist which have the function of
depriving infectivity of pathogens and activity of toxins. Antibody-mediated
neutralization
can be achieved by binding of an antigenic variable region to an antigen, or
can require
complement mediation. For example, in some cases, anti-viral antibodies
require
complement mediation in order to deprive a virus of its infectivity. Fc
regions are essential
to the participation of complements. Thus, such antibodies comprise effector
function that
requires Fc for neutralizing viruses and cells.
The present invention is based in part on the finding that anti-REG4
antibodies and
non-antibody binding proteins specifically bind to REG4, and then neutralize
REG4 activity
promoting cell proliferation, particularly in cancer cells where proliferation
is mediated by
abnormally high REG4 expression or intracellular signalling.
The terms "bind(s) specifically" or "specifically bind(s)" or "attached" or
"attaching"
in the context of antibodies or non-antibody binding proteins refers to the
preferential
association of an agent or ligand, in whole or part, with a target epitope
(e.g. REG4) that binds
or competes with another agent or ligand for binding to REG4 expressed on a
cell or tissue.
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It is, of course, recognized that a certain degree of non-specific interaction
may occur between
an antibody and a non-target epitope. Nevertheless, specific binding, can be
distinguished as
mediated through specific recognition of the target epitope. Typically
specific binding
results in a much stronger association between the delivered molecule and an
entity (e.g., an
assay well or a cell) bearing the target epitope than between the bound
antibody and an entity
(e.g., an assay well or a cell) lacking the target epitope. Specific binding
typically results in
at least about a 2-fold increase over background, preferably greater than
about 10-fold and
most preferably greater than 100-fold increase in amount of bound agent or
ligand (per unit
time) to a cell or tissue bearing the target epitope (i.e. REG4) as compared
to a cell or tissue
lacking the target epitope. Specific binding between two entities generally
means an affinity
of at least 106 M 1. Affinities greater than 1081Vr1 or greater are preferred.
Specific
binding can be determined for nucleic acid as well as protein agents and
ligands. Specific
binding for nucleic acid agents can be determined using any assay known in the
art, including
but not limited to northern blots, gel shift assays and in situ hybridization.
Specific binding
for protein agents and ligands can be determined using any binding assay known
in the art,
including but not limited to gel electrophoresis, western blot, ELISA, flow
cytometry, and
immunohi stochemi stry.
The present invention also relates to methods for suppressing cell growth of
REG4-
expressing cells, which comprise the following steps:
1) contacting the REG4-expressing cells with anti-REG4 antibodies or anti-REG4
non-antibody binding proteins, and
2) neutralizing the cell proliferation activity of REG4.
In the methods or pharmaceutical compositions of the present invention, any
REG4-
expressing cell can be suppressed. For example, pancreatic cancer cells are
preferable as the
REG4-expressing cells of the present invention. Of these, pancreatic carcinoma
or cells are
preferable.
Cells and antibodies (or non-antibody binding proteins) can be contacted itz
vivo or
in vitro. When targeting in vivo cancer cells as the REG4-expressing cells,
the methods of
the present invention are in fact therapeutic methods or preventative methods
for cancers.
Specifically, the present invention provides therapeutic methods for cancers
which comprise
the following steps:
1) administering an antibody or non-antibody binding protein that specifically
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binds REG4 to a cancer patient, and
2) suppressing cancer cell growth using the function of the antibody or non-
antibody binding protein bound to REG4, wherein the function is neutralizing
the cell proliferation activity of REG4.
In the present invention, neutralizing function of the anti-REG4 antibody or
non-
antibody binding protein refers to inhibition of REG4 activity for stimulating
cell proliferation
of REG4 expression cells. For instance, the present inventors confirm that
REG4 functions
as an autocrine or paracrine growth factor and mediate Akt signaling pathways.
Accordingly,
in the preferable embodiments of the present invention, anti-REG4 antibodies
or non-antibody
binding proteins shut down the REG4 autocrine/paracrine pathway and block the
subsequent
Akt phosphorylation. The present inventors also confirmed that antibodies
binding REG4
effectively suppress the cell proliferation of REG4-expressing cells, in
particular, pancreatic
cancer cells using neutralizing function. The present inventors further
confirmed that REG4
is highly expressed in pancreatic cancer cells, with a high probability. In
addition, REG4
expression levels in normal tissues are low. Putting this information
together, methods of
pancreatic cancer therapy where anti-REG4 antibody is administered can be
effective, with
little danger of side effects.
However, the antibodies and non-antibody binding proteins of the present
invention
are not limited so long as they comprise a desired neutralizing function.
Variants, analogs or
derivatives of the Fc portion may be constructed by, for example, making
various
substitutions of residues or sequences.
Variant (or analog) polypeptides include insertion variants, wherein one or
more
amino acid residues supplement an Fc amino acid sequence. Insertions may be
located at
either or both termini of the protein, or may be positioned within internal
regions of the Fc
amino acid sequence. Insertional variants with additional residues at either
or both termini
can include, for example, fusion proteins and proteins including amino acid
tags or labels.
For example, the Fc molecule may optionally contain an N-terminal Met,
especially when the
molecule is expressed recombinantly in a bacterial cell such as E. coli.
In Fc deletion variants, one or more amino acid residues in an Fe polypeptide
are
removed. Deletions can be effected at one or both terinini of the Fc
polypeptide, or with
removal of one or more residues within the Fc amino acid sequence. Deletion
variants,
therefore, include all fragments of an Fc polypeptide sequence.
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In Fc substitution variants, one or more amino acid residues of an Fc
polypeptide
are removed and replaced with alternative residues. In one aspect, the
substitutions are
conservative in nature, however, the invention embraces substitutions that are
non-
conservative.
Preferably, the parent polypeptide Fc region is a human Fc region, e.g., a
native
sequence human Fc region human IgGl (A and non-A allotypes) or human IgG3 Fc
region.
In one embodiment, the variant with improved ADCC mediates ADCC substantially
more
effectively than an antibody with a native sequence IgGl or IgG3 Fc region and
the antigen-
binding region of the variant. Preferably, the variant comprises, or consists
essentially of,
substitutions of two or three of the residues at positions 298, 333 and 334 of
the Fc region.
The numbering of the residues in an immunoglobulin heavy chain is that of the
EU index as in
Kabat et al., (saspra), expressly incorporated herein by reference. Most
preferably, residues
at positions 298, 333 and 334 are substituted, (e.g., with alanine residues).
Moreover, in
order to generate the Fc region variant with improved ADCC activity, one will
generally
engineer an Fc region variant with improved binding affinity for FcyRIII,
which is thought to
be an important FcR for mediating ADCC. For example, one may introduce an
amino acid
modification (e.g., an insertion, a deletion, or a substitution) into the
parent Fc region at any
one or more of amino acid positions 256, 290, 298, 312, 326, 330, 333, 334,
360, 378 or 430
to generate such a variant. The variant with improved binding affinity for
FayRIII may
further have reduced binding affinity for FcyRII; especially reduced affinity
for the inhibiting
Fc7RIIB receptor.
In any event, any variant amino acid insertions, deletions and/or
substitutions (e.g.,
from 1-50 amino acids, preferably, from 1-25 amino acids, more preferably,
from 1-10 amino
acids) are contemplated and are within the scope of the present invention.
Conservative
amino acid substitutions will generally be preferred. Furthermore, alterations
may be in the
form of altered amino acids, such as peptidomimetics or D-amino acids.
Therefore, human-derived antibodies belonging to these classes are preferable
in the
present invention. Human antibodies can be acquired using antibody-producing
cells
harvested from humans, or chimeric animals transplanted with human antibody
genes (Ishida
I, etal., (2002) Cloning and Stem Cells., 4: 91-102.).
Furthermore, antibody Fc regions can link with arbitrary variable regions.
Specifically, chimeric antibodies wherein the variable regions of different
animal species are
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bound to human constant regions are known. Alternatively, a human-human
chimeric
antibody can also be acquired by binding human-derived variable regions to
arbitrary constant
regions. In addition, CDR graft technology, where complementarity determining
regions
(CDRs) composing human antibody variable regions are replaced with CDRs of
heterologous
antibodies, is also known ("Immunoglobulin genes", Academic Press (London),
pp260-274,
1989; Roguska MA, et al., (1994) Proc. Natl. Acad. Sci. USA., 91: 969-73.).
By replacing CDRs, antibody binding specificity is replaced. That is, human
REG4 will be recognized by humanized antibodies in which the CDR of human REG4-
binding antibodies has been transferred. The transferred antibodies can also
be called
humanized antibodies. Antibodies thus-obtained and equipped with an Fc region
essential to
effector function can be used as the antibodies of the present invention,
regardless of the
origin of their variable regions. For example, antibodies comprising a human
IgG Fc are
preferable in the present invention, even if their variable regions comprise
an amino acid
sequence derived from an immunoglobulin of another class or another species.
Alternatively, antibody fragment that lacks Fc region may be used so long as
they
comprise a desired neutralizing function. For example, in the present
invention, Fv, Fab,
Fab', F(ab')2, or other antigen-binding subsequences of antibodies may also be
used as
antibody. In some embodiments, an agent to enhance the neutralizing effect may
be
conjugated with antibody, or fragment thereof.
VH and VL domains of antibodies of the present invention each comprise three
CDRs designated as CDR1, CDR2, and CDR3 separated by framework regions. Amino
acid
sequences of the CDRs are not particularly limited as long as the antibody can
specifically
bind to REG4. Preferred examples of CDR amino acid sequences include:
VH CDRI : SYW1V1pI (SEQ ID NO: 20),
VH CDR2 : NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3 : GGLWLRVDY (SEQ ID NO: 22),
VL CDR1 : SASSSVSYMH (SEQ IDNO: 23),
VL CDR2 : DTSKLAS (SEQ ID NO: 24),
VL CDR3 : QQWSSNPF (SEQ ID NO: 25)
In a more preferred embodiment, VH comprises the amino acid sequence of SEQ
ID NO: 18, and VL comprises the amino acid sequence of SEQ ID NO: 19. In some
embodiments, the VH comprises an amino acid sequence having at least about
90%, 95%,
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98% sequence identity to the full-length of SEQ ID NO: 18, and the VL
comprises an amino
acid sequence having at least about 90%, 95%, 98% sequence identity to the
full-length of
SEQ ID NO: 19. Sequence identity can be measured using default settings of
available
software well known in the art, for example, BLAST or ALIGN.
In the present invention, the antibodies may be monoclonal antibodies or
polyclonal
antibodies. Even when administering to humans, human polyclonal antibodies can
be
derived using the above-mentioned animals transferred with a human antibody
gene.
Alternatively, immunoglobulins which have been constructed using genetic
engineering
techniques, such as humanized antibodies, human-non-human chimeric antibodies,
and
human-human chimeric antibodies, can be used. Furthermore, methods for
obtaining human
monoclonal antibodies by cloning human antibody-producing cells are also
known.
REG4, or a fragment comprising its partial peptide, can be used as immunogens
to
obtain the antibodies. In the present invention, REG4 can be derived from any
species,
preferably from a mammal such as a human, mouse, or rat, and more preferably
from a human.
The human REG4 nucleotide sequence and amino acid sequence are known. The cDNA
nucleotide sequence of REG4 is described in SEQ ID NO: 1 and the amino acid
sequences
coded by that nucleotide sequence is described in SEQ ID NO: 2 (GenBank
Accession No.
AY126670). One skilled in the art can routinely isolate genes comprising the
provided
nucleotide sequence, preparing a fragment of the sequence as required, and
obtain a protein
comprising the target amino acid sequence.
For example, the gene coding the REG4 protein or its fragment can be inserted
into
a known expression vector, and used to transform host cells. The desired
protein, or its
fragment, can be collected from inside or outside host cells using arbitrary
and standard
methods, and can also be used as an antigen. In addition, proteins, their
lysates, and
chemically-synthesized proteins can be used as antigens. Furthermore, cells
expressing the
REG4 protein or a fragment thereof can themselves be used as immunogens.
When using a peptide fragment as the REG4 immunogen, it is particularly
preferable to select an amino acid sequence wllich comprises a region
predicted to be an
extra-cellular domain. The existence of a signal peptide is predicted from
positions 1 to 25
on the N-terminal of REG4. Thus, for example, a region other than the N-
terminal signal
peptide (25 amino acid residues) is preferred as the immunogen for obtaining
the antibodies
of the present invention. That is to say, antibodies that bind to REG4 extra-
cellular domains
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are preferred as the antibodies of the present invention.
Therefore, preferable antibodies in the present invention are antibodies
equipped
with an Fc essential to effector function, and a variable region that can bind
to an extracellular
REG4 domain. When aiming for administration to humans, it is preferable to be
equipped
with an IgG Fc.
Any mammal can be immunized with such an antigen. However, it is preferable to
consider compatibility with parent cells used in cell fusion. Generally,
rodents, lagomorphs,
or primates are used.
Rodents include, for example, mice, rats, and hamsters. Lagomorphs include,
for
example, rabbits. Primates include, for example, catarrhine (old world)
monlceys such as
Macaca fasciculaYis, Macaca naulatta, Sacred baboons, and chimpanzees.
Methods for immunizing animals with antigens are well known in the field.
Intraperitoneal or subcutaneous antigen injections are standard methods for
immunizing
mammals. Specifically, antigens can be diluted and suspended in an appropriate
amount of
phosphate buffered saline (PBS), physiological saline, or so on. As desired,
antigen
suspensions can be mixed with an appropriate amount of a standard adjuvant
such as Freund's
complete adjuvant, and administered to mammals after emulsification.
Subsequently, it is
preferable that antigens mixed with an appropriate amount of Freund's
incomplete adjuvant
are administered in multiple doses every four to 21 days. An appropriate
carrier can also be
used for immunization. After carrying out immunization as outlined above,
standard
methods can be used to examine serum for an increase in the desired antibody
level.
Polyclonal antibodies against the REG4 protein can be prepared from immunized
mammals whose serum has been investigated for an increase in the desired
antibodies. This
can be achieved by collecting blood from these animals, or by using an
arbitrary, usual
method to isolate serum from their blood. Polyclonal antibodies comprise serum
that
comprises polyclonal antibodies, and fractions that comprise polyclonal
antibodies which can
be isolated from serum. IgG and IgM can be prepared from fractions that
recognize REG4
protein by using, for example, an affinity column coupled to REG4 protein, and
then further
purifying this fraction using protein A or protein G columns. In the present
invention,
antiserum can be used as is as polyclonal antibodies. Alternatively, purified
IgG, IgM, or
such can also be used.
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To prepare monoclonal antibodies, immunocytes are collected from mammals
immunized with antigens, investigated for the increase of the desired antibody
level in serum
(as above), and applied in cell fusion. Immunocytes for use in cell fusion
preferably come
from the spleen. Other preferred parent cells for fusion with the above
immunogens include,
for example, mammalian myeloma cells, and more preferably, myeloma cells that
have
acquired properties for selection of fusion cells by pharmaceutical agents.
The above immunocytes and myeloma cells can be fused using known methods, for
example the methods of Milstein et al. (Galfre, G. and Milstein, C., (1981)
Methods.
Enzymol. : 73, 3-46.).
Hybridomas produced by cell fusion can be selected by culturing in a standard
selective medium such as HAT medium (medium comprising hypoxanthine,
aminopterin, and
thymidine). Cell culture in HAT medium is usually continued for several days
to several
weeks, a period sufficient enough to kill all cells other than the desired
hybridomas (unfused
cells). Standard limiting dilutions are then carried out, and hybridoma cells
that produce the
desired antibodies are screened and cloned.
Non-human animals can be immunized with antigens for preparing hybridomas in
the above method. In addition, human lymphocytes from cells infected witli EB
virus or
such, can be immunized in vitro using proteins, cells expressing proteins, or
suspensions of
the same. The immunized lymphocytes are then fused with human-derived myeloma
cells
able to divide unlimitedly (U266 and so on), thus obtaining hybridomas that
produce the
desired human antibodies which can bind the protein (Unexamined Published
Japanese Patent
Application No. (JP-A) Sho 63-17688).
The obtained liybridomas are then transplanted to mice abdominal cavities, and
ascites are extracted. The obtained monoclonal antibodies can be purified
using, for
example, ammonium sulfate precipitation, protein A or protein G columns, DEA.E
ion
exchange chromatography, or affinity columns coupled to the proteins of the
present
invention. The antibodies of the present invention can be used not only in
purifying and
detecting the proteins of the present invention, but also as candidates for
agonists and
antagonists of the proteins of the present invention. These antibodies can
also be applied to
antibody therapies for diseases related to the proteins of the present
invention. When the
obtained antibodies are administered to human bodies (antibody therapy), human
antibodies
or humanized antibodies are preferred due to their low immunogenicity.
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For example, transgenic animals comprising a repertoire of human antibody
genes
can be immunized with antigens selected from proteins, protein-expressing
cells, or
suspensions of the same. Antibody-producing cells are then recovered from the
animals,
fused with myeloma cells to yield hybridomas, and anti-protein human
antibodies can be
prepared from these hybridomas (see International Publication No. 92-03918, 94-
02602, 94-
25585, 96-33735, and 96-34096).
Alternatively, immunocytes such as immunized lymphocytes that produce
antibodies, can be immortalized using cancer genes, and used to prepare
monoclonal
antibodies.
Monoclonal antibodies obtained in this way can be prepared using methods of
genetic engineering (for example, see Borrebaeck, C.A.K. and Larrick, J.W.,
(1990)
Therapeutic Monoclonal Antibodies, MacMillan Publishers, UK). For example,
recombinant antibodies can be prepared by cloning DNAs that encode antibodies
from
immunocytes such as hybridomas or immunized lymphocytes that produce
antibodies; then
inserting these DNAs into appropriate vectors; and transforming these into
host cells.
Recombinant antibodies prepared as above can also be used in the present
invention.
The antibodies can be modified by binding with a variety of molecules such as
polyethylene glycols (PEGs). Antibodies modified in this way can also be used
in the
present invention. Modified antibodies can be obtained by chemically modifying
antibodies.
These kinds of modification methods are conventional to those skilled in the
art. The
antibodies can also be modified by other proteins. Antibodies modified by
protein
molecules can be produced using genetic engineering. That is, target proteins
can be
expressed by fusing antibody genes with genes that code for modification
proteins.
Alternatively, such antibodies can be obtained as chimeric antibodies which
comprise a non-human antibody-derived variable region and a human antibody-
derived
constant region, or as humanized antibodies which comprise a non-human
antibody-derived
complementarity determining region (CDR), a human antibody-derived framework
region
(FR), and a constant region. Such antibodies can be produced using lcnown
methods.
The standard techniques of molecular biology may be used to prepare DNA
sequences coding for the chimeric and CDR-grafted products. Genes encoding the
CDR of
an antibody of interest are prepared, for example, by using the polymerase
chain reaction
(PCR) to synthesize the variable region from RNA of antibody-producing cells
(see, for
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example, Larrick et al., "Methods: a Companion to Methods in Enzymology", vol.
2: page
106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies" in
Monoclonal Antibodies: Production, Engineering and Clinical Application;
Ritter et al. (eds.),
page 166 (Cambridge University Press, 1995), and Ward et al., "Genetic
Manipulation and
Expression of Antibodies" in Monoclonal Antibodies: Principles and
Applications; Birch et al.
(eds.), page 137 (Wiley-Liss, Inc., 1995)).
DNA sequences coding for the chimeric and CDR-grafted products may be
synthesised completely or in part using oligonucleotide synthesis techniques.
Site-directed
mutagenesis and polymerase chain reaction techniques may be used as
appropriate. For
example, oligonucleotide directed synthesis as described by Jones et al.,
(1986)
Nature.;321:522-5 may be used. Also oligonucleotide directed mutagenesis of a
pre-exising
variable region as, for example, described by Verhoeyen et al., (1988)
Science.;239:1534-6 or
Riechmann et al., (supra) may be used. Also enzymatic filling in of gapped
oligonucleotides
using T4 DNA polymerase as, for example, described by Queen et al., (1989)
Proc Natl Acad
Sci USA.;86:10029-33; PCT Publication WO 90/07861 may be used.
Any suitable host cell/vector system may be used for expression of the DNA
sequences coding for the CDR-grafted heavy and light chains. Bacterial, e.g.,
E. coli, and
other microbial systems may be used, in particular for expression of antibody
fragments such
as FAb and (Fab')2 fragments, and especially Fv fragments and single-chain
antibody
fragments, e.g., single-chain Fvs. Eucaryotic, e.g., mammalian, host cell
expression systems
may be used, in particular, for production of larger CDR-grafted antibody
products, including
coinplete antibody molecules. Suitable mammalian host cells include CHO cells
and
myeloma or hybridoma cell lines.
Antibodies obtained as above can be purified until uniform. For example,
antibodies can be purified or separated according to general methods used for
purifying and
separating proteins. For example, antibodies can be separated and isolated
using
appropriately selected combinations of column chromatography, comprising but
not limited to
affinity chromatography, filtration, ultrafiltration, salt precipitation,
dialysis, SDS
polyacrylamide gel electrophoresis, isoelectric focusing, and so on
(Antibodies : A
Laboratory Manual, Harlow and David, Lane (edit.), Cold Spring Harbor
Laboratory, 1988).
Protein A columns and Protein G columns can be used as affinity columns.
Exemplary protein A columns in use include Hyper D, POROS, and Sepharose FF
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(Pharmacia).
Exemplary chromatography (excluding affinity chromatography) include ion
exchange chromatography, hydrophobic chromatography, gel filtration, reverse
phase
chromatography, and adsorption chromatography ("Strategies for Protein
Purification and
Characterization:A Laboratory Course Manual" Daniel R. Marshak et al., (1996)
Cold Spring
Harbor Laboratory Press.). The chromatography can be performed according to
the
procedure of liquid phase chromatographies such as HPLC or FPLC.
For example, the antigen-binding activity of the antibodies of the present
invention
can be measured by using absorbance measurements, enzyme linked immunosorbent
assays
(ELISA), enzyme immunoassays (EIA), radioimmunoassays (RIA) and/or
immunofluorescence methods. In ELISA, an antibody of the present invention is
immobilized on a plate, a protein of the present invention is added to the
plate, and then a
sample comprising the desired antibody such as the culture supernatant of
cells that produce
the antibody or purified antibody is added. A secondary antibody that
recognizes the
primary antibody and has been tagged with an enzyme such as alkaline
phosphatase is then
added, and the plate is incubated. After washing, an enzyme substrate such as
p-nitrophenyl
phosphate is added to the plate, absorbance is measured, and the antigen-
binding activity of
the samples is evaluated. Protein fragments (C-terminal or N-terminal
fragments, and such)
can be used in the same way as proteins. The binding activity of the
antibodies can be
evaluated using BIAcore (Pharmacia).
Furthermore, one or more anti-REG4 antibodies which inhibit REG4 activity are
used for the methods and compositions of the present invention. In the present
invention, a
preferable anti-REG4 antibody neutralizes REG4 activity to promote cell
proliferation of
pancreatic cancer. The neutralizing function of anti-REG4 antibody can be
evaluated in
vitro or in vivo. For instance, the neutralizing function of anti-REG4
antibody can be
estimated by observing the effect of the antibody on proliferation of REG4
expressing cells.
Specifically, the neutralizing function would be acknowledged, when the
antibody detectably
suppresses the cell proliferation as measured using any method known in the
art.
Alternatively, such function also be confirmed by inhibition of Alct signaling
pathway
(Sekikawa A, et al. Gastroenterology 2005; 128: 642-53., Bishnupuri KS, et al.
Gastroenterology 2006; 130: 137-49.).
In the present invention, anti-REG4 antibodies and non-antibody binding
proteins
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can be administered to humans or other animals as pharmaceutical agents. In
the present
invention, animals other than humans to which the antibodies can be
administered include
mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows,
monkeys, baboons,
and chimpanzees. The antibodies and non-antibody binding proteins can be
directly
administered to subjects, and in addition, can be formulated into dosage forms
using known
pharmaceutical formulation methods. For example, depending on requirements,
they can be
parenterally administered in an injectable form such as a sterile solution or
suspension with
water or other arbitrary pharmaceutically acceptable fluid. For example, this
kind of
compounds can be mixed with acceptable carriers or solvents, specifically
sterile water,
physiological saline, vegetable oils, emulsifiers, suspension agents,
surfactants, stabilizers,
flavoring agents, excipients, solvents, preservatives, binding agents and the
like, into a
generally accepted unit dosage essential for use as a pharmaceutical agent.
Other isotonic solutions comprising physiological saline, glucose, and
adjuvants
(such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride) can be used
as the
injectable aqueous solution. They can also be used with appropriate
solubilizers such as
alcohols, specifically ethanols and polyalcohols (for example, propylene
glycols and
polyetliylene glycol), and non-ionic surfactants (for example Polysorbate 80TM
or HCO-50).
Sesame oils or soybean oils can be used as an oleaginous solution, and benzyl
benzoate or benzyl alcohols can be used with them as a solubilizer. Buffer
solutions
(phosphate buffers, sodium acetate buffers, or so on), analgesics (procaine
hydrochloride or
such), stabilizers (benzyl alcohol, phenols, or so on), and antioxidants can
be used in the
formulation. The prepared injections can be packaged into appropriate ampules.
In the present invention, the anti-REG4 antibodies and non-antibody binding
proteins
can be administered to patients, for example, intraarterially, intravenously,
percutaneously,
intranasally, transbronchially, locally, or intramuscularly. Intravascular
(intravenous)
administration by drip or injection is an example of a general method for
systematic
administration of antibodies to pancreatic cancer patients. In addition,
methods in which an
intraarterial catheter is inserted near a vein that supplies nutrients to
cancer cells to locally
inject anti-cancer agents such as antibody agents are effective as local
control therapies for
metastatic focuses as well as primary focuses of pancreatic cancer.
Although dosage and administration methods vary according to patient body
weight and age, and administration method, these can be routinely selected by
one skilled in
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the art. In addition, DNA encoding an antibody can be inserted into a vector
for gene
therapy, and the vector can be administered for therapy. Dosage and
administration methods
vary according to patient body weight, age, and condition, however, one
skilled in the art can
select these appropriately. Usually a lower dose is administered at first and
then
incrementally increased until an efficacious effect is achieved without
undesirable side effects.
Anti-REG4 antibodies and non-antibody binding proteins can be administered to
living bodies in an amount such that neutralizing function against REG4 can be
confirmed.
For example, although there is a certain amount of difference depending on
symptoms, anti-
REG4 antibody dosage is 0.1 mg to 250 mg/kg per day. Usually, the dosage for
an adult (of
weight 60 kg) is 5 mg to 17.5 g/day, preferably 5 mg to 10 g/day, and more
preferably 100 mg
to 3 g/day. The dosage schedule is from one to ten times over a two to ten day
interval, and
for example, progress is observed after a three to six times administration.
Alternatively, nucleic acids comprising sequences encoding antibodies, non-
antibody binding proteins, or functional derivatives thereof, are administered
to treat or
prevent diseases associated with REG4-expressing cells, such as pancreatic
cancer including
PDAC, by way of gene therapy. Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible nucleic acid. In
this embodiment
of the invention, the nucleic acids produce their encoded antibody or antibody
fragment that
mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the
present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
(1993) Clin.
Pharm.;12:488-505; Wu and Wu, (1991) Biotherapy.;3:87-95; Tolstoshev, (1993)
Ann Rev
Pharmacol Toxicol.;32:573-96; Mulligan, (1993) Science.;260:926-32; Morgan and
Anderson,
(1993) Ann Rev Biochem.;62:191-217; Trends Biotechnol.;11(5):155-215. Methods
commonly known in the art of recombinant DNA technology which can be used are
described
in Ausubel et al. (eds.), Current Protocols in Molecular Biology, Johii Wiley
& Sons, NY
(1993); K riegler, Gene Transfer and Expression, A Laboratory Manual,
Stoclctotl Press, NY
(1990).
In a preferred aspect, a composition of the invention comprises nucleic acids
encoding
an antibody, or a non-antibody binding protein, said nucleic acids being part
of an expression
vector that expresses the antibody or fragments or chimeric proteins or heavy
or light chains
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thereof in a suitable host. In particular, such nucleic acids have promoters,
preferably
heterologous promoters, operably linked to the antibody coding region, said
promoter being
inducible or constitutive, and, optionally, tissue-specific. In another
particular embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other desired
sequences are flanked by regions that promote homologous recombination at a
desired site in
the genome, thus providing for intrachromosomal expression of the antibody
encoding nucleic
acids (Koller and Smithies, (1989) Proc Natl Acad Sci USA.;86:8932-5; Zijlstra
et al., (1989)
Nature.;342:435-8). In specific embodiments, the expressed antibody molecule
is a single
chain antibody; alternatively, the nucleic acid sequences include sequences
encoding both the
heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a subject may be either direct, in which
case the
subject is directly exposed to the nucleic acid or nucleic acid-carrying
vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in vitro, then
transplanted into
the subject. These two approaches are known, respectively, as in vivo or ex
vivo gene
therapy.
In a specific embodiment, the nucleic acid sequences are directly administered
in vivo,
where it is expressed to produce the encoded product. This can be accomplished
by any of
numerous methods known in the art, e.g., by constructing them as part of an
appropriate
nucleic acid expression vector and administering it so that they become
intracellular, e.g., by
infection using defective or attenuated retrovirals or other viral vectors
(see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of microparticle
bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or
transfecting agents, encapsulation in liposomes, microparticles, or
microcapsules, or by
administering them in linkage to a peptide which is known to enter the
nucleus, by
administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see, e.g., Wu
and Wu, (1987) J Biol Chem.;262:4429-32) (which can be used to target cell
types
specifically expressing the receptors), etc.
In another embodiment, nucleic acid-ligand complexes can be formed in which
the
ligand coinprises a fusogenic viral peptide to disrupt endosomes, allowing the
nucleic acid to
avoid lysosomal degradation. In yet another embodiment, the nucleic acid can
be targeted in
vivo for cell specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT
Publications WO 92/06180, WO 92/22635, W092/20316, W093/14188 or WO 93/20221).
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Alternatively, the nucleic acid can be introduced intracellularly and
incorporated within host
cell DNA for expression, by homologous recombination (Koller and Smithies,
(1989) Proc
Natl Acad Sci USA.;86:8932-5; Zijlstra et al., (1989) Nature.;342:435-8).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding
an antibody of the invention or fragments thereof are used. For example, a
retroviral vector
can be used (see Miller et al., (1993) Methods Enzymol.;217:581-99). These
retroviral
vectors contain the components necessary for the correct packaging of the
viral genome and
integration into the host cell DNA. The nucleic acid sequences encoding the
antibody or
non-antibody binding proteins to be used in gene therapy are cloned into one
or more vectors,
which facilitates delivery of the gene into a subject. More detail about
retroviral vectors can
be found in Boesen et al., (1994) Biotherapy.;6:291-302, which describes the
use of a
retroviral vector to deliver the mdr 1 gene to hematopoietic stem cells in
order to make the
stem cells more resistant to chemotherapy. Other references illustrating the
use of retroviral
vectors in gene therapy are: Clowes et al., (1994) J Clin Invest.;93:644-51;
Keim et al.,
(1994) Blood.;83:1467-73; Salmons and Gunzberg, (1993) Hum Gene Ther.;4:129-
41;
Grossman and Wilson, (1993) Curr Opin Genet Dev.;3:110-4.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses
are especially attractive vehicles for delivering genes to respiratory
epithelia. Adenoviruses
naturally infect respiratory epithelia where they cause a mild disease. Other
targets for
adenovirus-based delivery systems are liver, the central nervous system,
endothelial cells, and
muscle. Adenoviruses have the advantage of being capable of infecting non-
dividing cells.
Kozarsky and Wilson, in (1993) Curr Opin Genet Dev.;3:499-503, present a
review of
adenovirus-based gene therapy. Bout et al., in (1994) Hum Gene Ther.;5:3-10,
demonstrates
the use of adenovirus vectors to transfer genes to the respiratory epithelia
of rhesus monkeys.
Other instances of the use of adenoviruses in gene therapy can be found in
Rosenfeld et al.,
(1991) Science.;252:431-4; Rosenfeld et al., (1992) Cell.;68:143-55;
Mastrangeli et al.,
(1993) J Clin Invest.;91:225-34; PCT Publication WO94/12649; Wang et al.,
(1995) Gene
Ther.;2:775-83. In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh
et al., (1993) Proc Soc Exp Biol Med.;204:289-300; U.S. Pat. No. 5,436,146).
Another approach to gene therapy involves transferring a gene to cells in
tissue culture
by such methods as electroporation, lipofection, calcium phosphate mediated
transfection, or
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viral infection. Usually, the method of transfer includes the transfer of a
selectable marker to
the cells. The cells are then placed under selection to isolate those cells
that have taken up
and are expressing the transferred gene. Those cells are then delivered to a
subject.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration
in vivo of the resulting recombinant cell. Such introduction can be carried
out by any
method known in the art, including but not limited to transfection,
electroporation,
microinjection, infection with a viral or bacteriophage vector containing the
nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated
gene transfer,
spheroplast fusion, etc. Numerous techniques are known in the art for the
introduction of
foreign genes into cells (see, e.g., Loeffler and Behr, (1993) Methods
Enzymol.;217:599-618;
Cotton et al., 1993, Methods Enzymol.;217:618-44; Cline MJ. Pharmacol Ther.
1985;29(1):69-92.) and may be used in accordance with the present invention,
provided that
the necessary developmental and physiological functions of the recipient cells
are not
disrupted. The technique should provide for the stable transfer of the nucleic
acid to the cell,
so that the nucleic acid is expressible by the cell and preferably heritable
and expressible by
its cell progeny.
The resulting recombinant cells can be delivered to a subject by various
methods
known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are
preferably administered intravenously. The amount of cells envisioned for use
depends on
the desired effect, patient state, etc., and can be determined by one skilled
in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes;
blood cells such as T
lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, granulocytes; various stem or progenitor cells, in particular
hematopoietic
stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord
blood, peripheral
blood, fetal liver, etc. In a preferred embodiment, the cell used for gene
therapy is
autologous to the subject.
In an embodiment in which recombinant cells are used in gene therapy, nucleic
acid sequences encoding an antibody or fragment thereof are introduced into
the cells such
that they are expressible by the cells or their progeny, and the recombinant
cells are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor
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cells are used. Any stem and/or progenitor cells which can be isolated and
maintained in
vitro can potentially be used in accordance with this embodiment of the
present invention (see
e.g., PCT Publication WO 94/08598; Stemple and Anderson, (1992) Cell.;71:973-
85;
Rheinwald. (1980) Methods Cell Biol.;21A:229-54; Pittelkow and Scott, (1986)
Mayo Clin
5 Proc.;61:771-7).
In a specific embodiment, the nucleic acid to be introduced for purposes of
gene
therapy comprises an inducible promoter operably linked to the coding region,
such that
expression of the nucleic acid is controllable by controlling the presence or
absence of the
appropriate inducer of transcription.
10 In addition, the present invention provides immunogenic compositions for
inducing
antibodies coinprising neutralizing functions against REG4, where the
compositions comprise
as an active ingredient REG4 or an immunologically active REG4 fragment, or a
DNA or cell
which can express the same. Alternatively, the present invention relates to
uses of REG4 or
an immunologically active REG4 fragment, or a DNA or cell which can express
the same in
15 the production of immunogenic compositions for inducing antibodies
comprising neutralizing
functions against REG4.
In the present invention, neutralizing of REG4 activity to promote cell
proliferation in
autocrine/paracrine manner can be achieved by the administration of anti-REG4
antibodies.
Thus, if REG4 antibodies can be induced in vivo, therapeutic effects
equivalent to the
20 antibody adininistration can be achieved. When administering immunogenic
compositions
comprising antigens, target antibodies can be induced in vivo. The immunogenic
coinpositions of the present invention thus are particularly useful in vaccine
therapy against
REG4-expressing cells. Thus, the immunogenic compositions of the present
invention are
effective as, for example, vaccine compositions for pancreatic cancer
therapies.
25 The immunogenic compositions of the present invention can comprise REG4 or
an
immunologically active REG4 fragment, as an active ingredient. An
immunologically active
REG4 fragment refers to a fragment that can induce anti- REG4 antibodies which
recognize
REG4 and comprise neutralizing function. Below, REG4 and the immunologically
active
REG4 fragment are described as immunogenic proteins. Whether a given fragment
induces
30 target antibodies can be determined by actually immunizing an animal, and
confirming the
activity of the induced antibodies. Antibody induction and the confirmation of
its activity
can be carried out, for example, using methods described in Examples.
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The immunogenic compositions of the present invention comprise
pharmaceutically
acceptable carriers as well as immunogenic proteins, the active ingredients.
If necessary, the
compositions can also be combined with an adjuvant. Killed tuberculosis
bacteria,
diphtheria toxoid, saponin and so on can be used as the adjuvant.
Alternatively, DNAs coding for the immunogenic proteins, or cells retaining
those
DNAs in an expressible state, can be used as the immunogenic compositions.
Methods for
using DNAs expressing the target antigen as immunogens, so-called DNA
vaccines, are well
known. DNA vaccines can be obtained by inserting a DNA encoding REG4 or its
fragment
iiito an appropriate expression vector.
Retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai
virus
vectors or such can be used as the vector. In addition, DNAs in which a DNA
encoding an
immunogenic protein is functionally connected downstream of a promoter can be
directly
introduced into cells as naked DNA, and then expressed. Naked DNA can be
encapsulated
in liposomes or viral envelope vectors and introduced into cells.
As noted above, the present invention provides methods for inducing antibodies
which
comprise neutralizing function against REG4, where the methods comprise the
step of
administering REG4, an immunologically active REG4 fragment, or DNA or cells
that can
express the same. The methods of the present invention induce antibodies that
comprise
neutralizing function that suppresses cell growth of REG4-expressing cells
such as pancreatic
cancers. As a result, therapeutic effects for pancreatic cancers and so on can
be obtained.
Dominant neizative protein that inhibits KIAA0101:
The present invention relates to inhibitory polypeptides that contain QKGIGEFF
(SEQ ID NO: 46). In some preferred embodiments, the inhibitory polypeptide
comprises
QKGIGEFF (SEQ ID NO: 46); a polypeptide functionally equivalent to the
polypeptide; or
polynucleotide encoding those polypeptides, wherein the polypeptide lacks the
biological
function of a peptide consisting of SEQ ID NO: 40. The amino acid sequence set
forth in
SEQ IDNO: 40 is disclosed in WO2004/31412. It has been known that cancer cell
proliferation can be controlled by inhibiting the expression of the amino acid
sequence.
However, it is a novel finding proved by the present inventors that a fragment
containing a
sequence with a specific mutation in the above amino acid sequence inhibits
the cancer cell
proliferation.
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The polypeptides comprising the selected amino acid sequence of the present
invention, can be of any length, so long as the polypeptide inhibits cancer
cell proliferation.
Specifically, the length of the amino acid sequence may range from 8 to 70
residues, for
example, from 8 to 50, preferably from 8 to 30, more specifically from 8 to
20, further more
specifically from 8 to 16 residues. For example, the amino acid sequence
VRPTPKWQKGIGEFFRLSPK/SEQ ID NO. 44 is preferable as the above-described
selected
amino acid sequence. Therefore, a polypeptide comprising or consisting of the
amino acid
sequence TPKWQKGIGEFFRLSP/SEQ ID NO. 45 is a preferred example of the
polypeptides in the present invention. The polypeptides of the present
invention, which are
characterized by containing the amino acid sequence QKGIGEFF/SEQ ID NO: 46,
may also
be referred to as "PIP binding motif (PIP box)".
The polypeptides of the present invention may contain two or more "selected
amino
acid sequences". The two or more "selected amino acid sequences" may be the
same or
different amino acid sequences. Furthermore, the "selected amino acid
sequences" can be
linked directly. Alternatively, they may be disposed with any intervening
sequences among
them.
Furthermore, the present invention relates to polypeptides homologous (i.e.,
share
sequence identity) to the QKGIGEFF/SEQ ID NO: 46 polypeptide specifically
disclosed here.
In the present invention, polypeptides homologous to the QKGIGEFF/SEQ ID NO:
46
polypeptide are those which contain any mutations selected from addition,
deletion,
substitution and insertion of one or several amino acid residues and are
functionally
equivalent to the QKGIGEFF/SEQ ID NO: 46 polypeptide. The phrase "functionally
equivalent to the QKGIGEFF/SEQ ID NO: 46 polypeptide" refers to having the
function to
inhibit the binding of KIAA0101 to PCNA. The QKGIGEFF/SEQ IDNO: 46 sequence is
preferably conserved in the amino acid sequences constituting polypeptides
functionally
equivalent to QKGIGEFF/SEQ ID NO: 46 polypeptide. Therefore, polypeptides
functionally equivalent to the QKGIGEFF/SEQ ID NO: 46 peptide in the present
invention
preferably have amino acid mutations in sites other than the QKGIGEFF/SEQ ID
NO: 46
sequence. Amino acid sequences of polypeptides functionally equivalent to the
QKGIGEFF/SEQ ID NO: 46 peptide in the present invention conserve the
QKGIGEFF/SEQ
ID NO: 46 sequence, and have 60% or higher, usually 70% or higher, preferably
80% or
higher, more preferably 90% or higher, or 95% or higher, and further more
preferably 98% or
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higher homology to a "selected amino acid sequence". Amino acid sequence
homology can
be determined using algorithms well known in the art, for example, BLAST or
ALIGN set to
their default settings.
Alternatively, the number of amino acids that may be mutated is not
particularly
restricted, so long as the QKGIGEFF/SEQ ID NO: 46 peptide activity is
maintained.
Generally, up to about 50 amino acids may be mutated, preferably up to about
30 amino acids,
more preferably up to about 10 amino acids, and even more preferably up to
about 3 amino
acids. Likewise, the site of mutation is not particularly restricted, so long
as the mutation
does not result in the disruption of the QKGIGEFF/SEQ ID NO: 46 peptide
activity.
In a preferred embodiment, the activity of the QKGIGEFF/SEQ ID NO: 46 peptide
comprises apoptosis inducing effect in a IKIAA0101 expressing cell, i.e.
pancreatic cancer cell,
prostatic cancer cell, breast cancer cell, and bladder cancer cell. Apoptosis
means cell death
caused by the cell itself and is sometimes referred to as programmed cell
death. Aggregation
of nuclear chromosome, fragmentation of nucleus, or condensation of cytoplasm
is observed
in a cell undergoing apoptosis. Methods for detecting apoptosis are well
known. For
instance, apoptosis may be confirmed by TUNEL staining (Terminal
deoxynucleotidyl
Transferase Biotin-dUTP Nick End Labeling; Gavrieli et al., (1992) J. Cell
Biol. 119: 493-501,
Mori et al., (1994) Anat. & Embryol. 190: 21-28). Alternatively, DNA ladder
assays,
Annexin V staining, caspase assay, electron microscopy, or observation of
conformational
alterations on nucleus or cell membrane may be used for detecting apoptosis.
Any
commercially available kits may be used for detecting these behaviors in cells
which are
induced by apoptosis. For example, such apoptosis detection kits may be
commercially
available from the following providers:
LabChem Inc.,
Promega,
BD Biosciences Pharmingen,
Calbiochem,
Takara Bio Company (CLONTECH Inc.),
CHEMICON International, Inc,
Medical & Biological Laboratories Co., Ltd. etc.
The polypeptides of the present invention can be chemically synthesized from
any
position based on selected amino acid sequences. Methods used in the ordinary
peptide
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chemistry can be used for the method of synthesizing polypeptides.
Specifically, the
methods include those described in the following documents and Japanese Patent
publications:
Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2,
Academic
Press Inc., New York, 1976;
Peputido gousei (Peptide Synthesis), Maruzen (Inc.), 1975;
Peputido gousei no kiso to jikken (Fundamental and Experimental Peptide
Synthesis),
Maruzen (Inc.), 1985;
lyakuhin no kaihatsu (Development of Pharmaceuticals), Sequel, Vol. 14:
Peputido
gousei (Peptide Synthesis), Hirokawa Shoten, 1991;
International Patent Publication W099/67288.
The polypeptides of the present invention can be also synthesized by known
genetic
engineering techniques. An example of genetic engineering techniques is as
follows.
Specifically, DNA encoding a desired peptide is introduced iiito an
appropriate host cell to
prepare a transformed cell. The polypeptides of the present invention can be
obtained by
recovering polypeptides produced by this transformed cell. Alternatively, a
desired
polypeptide can be synthesized with an in vitro translation system, in which
necessary
elements for protein synthesis are reconstituted in vitro.
When genetic engineering techniques are used, the polypeptide of the present
invention can be expressed as a fused protein with a peptide having a
different amino acid
sequence. A vector expressing a desired fusion protein can be obtained by
linking a
polynucleotide encoding the polypeptide of the present invention to a
polynucleotide
encoding a different peptide so that they are in the same reading frame, and
then introducing
the resulting nucleotide into an expression vector. The fusion protein is
expressed by
transforming an appropriate host with the resulting vector. Different peptides
to be used in
forming fusion proteins include the following peptides:
FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10),
6xHis consisting of six His (histidine) residues, 10xHis,
Influenza hemagglutinin (HA),
Human c-myc fragment,
VSV-GP fragment,
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p 18 HIV fragment,
T7-tag,
HSV-tag,
E-tag,
SV40T antigen fragment,
lcle tag,
a-tubulin fragment,
B-tag,
Protein C fragment,
GST (glutathione-S-transferase),
HA (Influenza hemagglutinin),
Immunoglobulin constant region,
P-galactosidase, and
MBP (maltose-binding protein).
The polypeptide of the present invention can be obtained by treating the
fusion
protein thus produced with an appropriate protease, and then recovering the
desired
polypeptide. To purify the polypeptide, the fusion protein is captured in
advance with
affinity chromatography that binds with the fusion protein, and then the
captured fusion
protein can be treated with a protease. With the protease treatment, the
desired polypeptide
is separated from affinity chromatography, and the desired polypeptide with
high purity is
recovered.
The polypeptides of the present invention include modified polypeptides. In
the
present invention, the term "modified" refers, for example, to binding with
other substances.
Accordingly, in the present invention, the polypeptides of the present
invention may further
comprise other substances such as cell-membrane permeable substance. The other
substances include organic compounds such as peptides, lipids, saccharides,
and various
naturally-occurring or synthetic polymers. The polypeptides of the present
invention may
have any modifications so long as the polypeptides retain the desired activity
of inhibiting the
binding of KIAA0101 to PCNA. In some embodiments, the inhibitory polypeptides
can
directly compete with KIAA0101 binding to PCNA. Modifications can also confer
additive
functions on the polypeptides of the invention. Examples of the additive
functions include
targetability, deliverability, and stabilization.
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Preferred examples of modifications in the present invention include, for
example,
the introduction of a cell-membrane permeable substance. Usually, the
intracellular
structure is cut off from the outside by the cell membrane. Therefore, it is
difficult to
efficiently introduce an extracellular substance into cells. Cell membrane
permeability can
be conferred on the polypeptides of the present invention by modifying the
polypeptides with
a cell-membrane permeable substance. As a result, by contacting the
polypeptide of the
present invention with a cell, the polypeptide can be delivered into the cell
to act thereon.
The "cell-membrane permeable substance" refers to a substance capable of
penetrating the mammalian cell membrane to enter the cytoplasm. For example, a
certain
liposome fuses with the cell membrane to release the content into the cell.
Meanwhile, a
certain type of polypeptide penetrates the cytoplasmic membrane of mammalian
cell to enter
the inside of the cell. For polypeptides having such a cell-entering activity,
cytoplasmic
membranes and such in the present invention are preferable as the substance.
Specifically,
the present invention includes polypeptides having the following general
formula.
[R]-[D];
wherein,
[R] represents a cell-membrane permeable substance; [D] represents a fragment
sequence
containing QKGIGEFFlSEQ ID NO: 46. In the above-described general formula, [R]
and
[D] can be linked directly or indirectly through a linker. Peptides, compounds
having
multiple functional groups, or such can be used as a linker. Specifically,
amino acid
sequences containing -GGG- can be used as a linker. Alternatively, a cell-
membrane
permeable substance and a polypeptide containing a selected sequence can be
bound to the
surface of a minute particle. [R] can be linked to any positions of [D].
Specifically, [R]
can be linked to the N terminal or C terminal of [D], or to a side chain of
amino acids
constituting [D]. Furthermore, more than one [R] molecule can be linked to one
molecule of
[D]. The [R] molecules can be introduced to different positions on the [D]
molecule.
Alternatively, [D] can be modified with a number of [R]s linked together.
For example, there have been reported a variety of naturally-occurring or
artificially
synthesized polypeptides having cell-meinbrane permeability (Joliot A. &
Prochiantz A., Nat
Cell Biol. 2004; 6: 189-96). All of these known cell-membrane permeable
substances can be
used for modifying polypeptides in the present invention. In the present
invention, for
example, any substance selected from the following group can be used as the
above-described
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cell-permeable substance:
poly-arginine; Matsushita et al., (2003) J. Neurosci.; 21, 6000-7.
[Tat / RKKRRQRRR] (SEQ ID NO: 47) Frankel et al., (1988) Cell 55,1189-93.
Green & Loewenstein (1988) Cell 55, 1179-88.
[Penetratin / RQIKIWFQNRRMKWKK] (SEQ ID NO: 48)
Derossi et al., (1994) J. Biol. Chem. 269, 10444-50.
[Buforin II / TRSSRAGLQFPVGRVHRLLRK] (SEQ ID NO: 49)
Park et al., (2000) Proc. Natl Acad. Sci. USA 97, 8245-50.
[Transportan / GWTLNSAGYLLGKINLKALAALAKKIL] (SEQ ID NO: 50)
Pooga et al., (1998) FASEB J. 12, 67-77.
[MAP (model amphipathic peptide) / KLALKLALKALKAALKLA] (SEQ ID NO:
51)
Oehlke et al., (1998) Biochim. Biophys. Acta. 1414, 127-39.
[K-FGF / AAVALLPAVLLALLAP] (SEQ ID NO: 52)
Lin et al., (1995) J. Biol. Chem. 270, 14255-8.
[Ku70 / VPMLK] (SEQ ID NO: 53)
Sawada et al., (2003) Nature Cell Biol. 5, 352-7.
[Ku70 / PMLKE] (SEQ ID NO: 61)
Sawada et al., (2003) Nature Cell Biol. 5, 352-7.
[Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP] (SEQ ID NO: 54)
Lundberg et al., (2002) Biochem. Biophys. Res. Commun. 299, 85-90.
[pVEC / LLIILRRRIRKQAHAHSK] (SEQ ID NO: 55)
Elmquist et al., (2001) Exp. Cell Res. 269, 237-44.
[Pep-1 / KETWWETWWTEWSQPKKKRKV] (SEQ ID NO: 56)
Morris et al., (2001) Nature Biotechnol. 19, 1173-6.
[SynB 1/ RGGRLSYSRRRFSTSTGR] (SEQ ID NO: 57)
Rousselle et al., (2000) Mol. Pharmacol. 57, 679-86.
[Pep-7 / SDLWEMIVIlVIVSLACQY] (SEQ ID NO: 58)
Gao et al., (2002) Bioorg. Med. Chem. 10, 4057-65.
[HN-1 / TSPLNIHNGQKL] (SEQ ID NO: 59)
Hong & Clayman (2000) Cancer Res. 60, 6551-6.
In the present invention, the poly-arginine, which is listed above as an
example of cell-
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membrane permeable substances, is constituted by any number of arginine
residues.
Specifically, for example, it is constituted by consecutive 5-20 arginine
residues. The
preferable number of arginine residues is 11 (SEQ ID NO: 60).
Pharmaceutical compositions comprising_OKGIGEFF/SEQ ID NO: 46
The polypeptides of the present invention inhibit proliferation of cancer
cells.
Therefore, the present invention provides therapeutic and/or preventive agents
for cancer
which comprise as an active ingredient a polypeptide which comprises
QKGIGEFF/SEQ ID
NO: 46; or a polynucleotide encoding the same. Alternatively, the present
invention relates
to methods for treating and/or preventing cancer comprising the step of
administering a
polypeptide of the present invention. Furthermore, the present invention
relates to the use of
the polypeptides of the present invention in manufacturing pharmaceutical
compositions for
treating and/or preventing cancer. Cancers which can be treated or prevented
by the present
invention are not limited, so long as expression of KIAA0101 is up-regulated
in the cancer
cells. For example, the polypeptides of the present invention are useful for
treating
pancreatic cancer, lung cancer, prostatic cancer, breast cancer, bladder
cancer, kidney cancer
or testicular tumors. Among them, pancreatic cancer is particularly preferable
as a target for
treatment or prevention in the present invention.
Alternatively, the inhibitory polypeptides of the present invention can be
used to
induce apoptosis of cancer cells. Therefore, the present invention provides
apoptosis
inducing agents for cells, which comprise as an active ingredient a
polypeptide which
comprises QKGIGEFF/SEQ ID NO: 46; or a polynucleotide encoding the same. The
apoptosis inducing agents of the present invention may be used for treating
cell proliferative
diseases such as cancer. Cancers which can be treated or prevented by the
present invention
are not limited, so long as expression of KIAA0101 is up-regulated in the
cancer cells. For
example, the polypeptides of the present invention are useful in treating
pancreatic cancer,
prostatic cancer, breast cancer, bladder cancer, lung cancer, kidney cancer or
testicular tumors.
Among them, pancreatic cancer is particularly preferable as a target for
treatment or
prevention in the present invention. Alternatively, the present invention
relates to methods
for inducing apoptosis of cells which comprise the step of administering the
polypeptides of
the present invention. Furthermore, the present invention relates to the use
of polypeptides
of the present invention in manufacturing pharmaceutical compositions for
inducing apoptosis
in cells.
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The inhibitory polypeptides of the present invention induce apoptosis in
KIAA0101-expressing cells such as pancreatic cancer. In the meantime, KIAA0101
expression has not been observed in most of normal organs. In some normal
organs, the
expression level of KIAA0101 is relatively low as compared with cancer
tissues.
Accordingly, the polypeptides of the present invention may induce apoptosis
specifically in
cancer cells.
When the polypeptides of the present invention are administered, as a prepared
pharmaceutical, to human and other mammals such as mouse, rat, guinea pig,
rabbit, cat, dog,
sheep, pig, cattle, monkey, baboon and chimpanzee for treating cancer or
inducing apoptosis
in cells, isolated compounds can be administered directly, or formulated into
an appropriate
dosage form using known methods for preparing pharmaceuticals. For example, if
necessary,
the pharmaceuticals can be orally administered as a sugar-coated tablet,
capsule, elixir, and
microcapsule, or alternatively parenterally administered in the injection form
that is a
sterilized solution or suspension with water or any other pharmaceutically
acceptable liquid.
For example, the compounds can be mixed with pharmacologically acceptable
carriers or
media, specifically sterilized water, physiological saline, plant oil,
emulsifier, suspending
agent, surfactant, stabilizer, corrigent, excipient, vehicle, preservative,
and binder, in a unit
dosage form necessary for producing a generally accepted pharmaceutical.
Depending on
the amount of active ingredient in these formulations, a suitable dose within
the specified
range can be determined.
Examples of additives that can be mixed in tablets and capsules are binders
such as
gelatin, corn starch, tragacanth gum, and gum arabic; media such as
crystalline cellulose;
swelling agents such as corn starch, gelatin, and alginic acid; lubricants
such as magnesium
stearate; sweetening agents such as sucrose, lactose or saccharine; and
corrigents such as
peppermiiit, wintergreen oil and cherry. When the unit dosage from is capsule,
liquid
carriers such as oil can be further included in the above-described
ingredients. Sterilized
mixture for injection can be formulated using media such as distilled water
for injection
according to the realization of usual pharmaceuticals.
Physiological saline, glucose, and other isotonic solutions containing
adjuvants such
as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an
aqueous
solution for injection. They can be used in combination with a suitable
solubilizer, for
example, alcohol, specifically ethanol and polyalcohols such as propylene
glycol and
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polyethylene glycol, non-ionic surfactants such as Polysorbate 8OTM and HCO-
50.
Sesame oil or soybean oil can be used as an oleaginous liquid, and also used
in
combination with benzyl benzoate or benzyl alcohol as a solubilizer.
Furthermore, they can
be further formulated with buffers such as phosphate buffer and sodium acetate
buffer;
analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol
and phenol; and
antioxidants. Injections thus prepared can be loaded into appropriate
ampoules.
Methods well-known to those skilled in the art can be used for administering
pharmaceutical compounds of the present invention to patients, for example, by
intraarterial,
intravenous, or subcutaneous injection, and similarly, by intranasal,
transtracheal,
intramuscular, or oral administration. Doses and administration methods are
varied
depending on the body weight and age of patients as well as administration
methods.
However, those skilled in the art can routinely select them. DNA encoding a
polypeptide of
the present invention can be inserted into a vector for the gene therapy, and
the vector can be
administered for treatment. Although doses and administration methods are
varied
depending on the body weight, age, and symptoms of patients, those skilled in
the art can
appropriately select them. For example, a dose of the compound which bind to
the
polypeptides of the present invention so as to regulate their activity is,
when orally
administered to a normal adult (body weight 60 kg), about 0.1 mg to about 100
mg/day,
preferably about 1.0 mg to about 50 mg/day, more preferably about 1.0 mg to
about 20
mg/day, although it is sliglltly varied depending on symptoms.
When the compound is parenterally administered to a normal adult (body weight
60
kg) in the injection form, it is convenient to intravenously inject a dose of
about 0.01 mg to
about 30 mg/day, preferably about 0.1 mg to about 20 mg/day, more preferably
about 0.1 mg
to about 10 mg/day, although it is slightly varied depending on patients,
target organs,
symptoms, and administration methods. Similarly, the compound can be
administered to
other animals in an amount converted from the dose for the body weight of 60
kg.
Pharmaceutical compositions:
Accordingly, the present invention includes medicaments and methods useful in
either
or both preventing and treating cancers. These medicaments and methods
comprise at least a
siRNA that inhibits expression of REG4 or KIAA0101, the antibody that
neutralizes the
activity of REG4, a polypeptide which comprises QKGIGEFF/SEQ ID NO: 46; a
polypeptide
functionally equivalent to the polypeptide; or polynucleotide encoding those
polypeptides of
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the present invention in an amount effective to achieve attenuation or arrest
of disease cell
proliferation. More specifically, in the context of the present invention, a
therapeutically
effective amount means an amount effective to prevent development of, or to
alleviate
existing symptoms of, the subject being treated.
Individuals to be treated with methods of the present invention include any
individual
afflicted with cancer, including, e.g., pancreatic cancer, prostatic cancer,
breast cancer, and
bladder cancer. Such an individual can be, for example, a vertebrate such as a
mammal,
including a human, dog, cat, horse, cow, or goat; or any other animal,
particularly a
commercially important animal or a domesticated animal. For purposes of the
present
invention, elevated expression of marker proteins refers to a mean cellular
marker protein
concentration for one or both marker proteins that is at least 10%, preferably
15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55% or more above normal mean cellular concentration
of the
marker protein(s).
In the context of the present invention, suitable pharmaceutical formulations
include
those suitable for oral, rectal, nasal, topical (including buccal and sub-
lingual), vaginal or
parenteral (including intramuscular, sub-cutaneous and intravenous)
administration, or for
administration by inhalation or insufflation. Preferably, administration is
intravenous. The
formulations are optionally packaged in discrete dosage units.
Pharmaceutical formulations suitable for oral administration include capsules,
cachets
or tablets, each containing a predetermined amount of active ingredient.
Suitable
formulations also include powders, granules, solutions, suspensions and
emulsions. The
active ingredient is optionally administered as a bolus electuary or paste.
Tablets and
capsules for oral administration may contain conventional excipients,, for
example, fillers
such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose
preparations such as
maize starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or
polyvinylpyrrolidone (PVP), binding agents, lubricants, and/or wetting agents.
If desired,
disintegrating agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
A tablet may be made by compression or molding, optionally with one or more
formulational ingredients. Compressed tablets may be prepared by compressing
in a suitable
machine the active ingredients in a free-flowing form, such as a powder or
granules,
optionally mixed with a binder, lubricant, inert diluent, lubricating, surface
active and/or
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dispersing agent. Molded tablets may be made by molding in a suitable machine
a mixture
of the powdered compound moistened with an inert liquid diluent. The tablets
may be
coated according to methods well known in the art. Oral fluid preparations may
be in the
form of, for example, aqueous or oily suspensions, solutions, emulsions,
syrups or elixirs, or
may be presented as a dry product for constitution with water or other
suitable vehicle before
use. Such liquid preparations may contain conventional additives such as
suspending agents,
emulsifying agents, non-aqueous vehicles (which may include edible oils), pH
maintaining
agents, and/or preservatives. The tablets may optionally be formulated so as
to provide slow
or controlled release of the active ingredient therein. A package of tablets
may contain one
tablet to be taken on each of the month.
Formulations suitable for parenteral administration include aqueous and non-
aqueous
sterile injection solutions, optionally contain anti-oxidants, buffers,
bacteriostats and solutes
which render the formulation isotonic with the blood of the intended
recipient; as well as
aqueous and non-aqueous sterile suspensions including suspending agents and/or
thickening
agents. The formulations may be presented in unit dose or multi-dose
containers, for
example as sealed ampoules and vials, and may be stored in a freeze-dried
(lyophilized)
condition requiring only the addition of the sterile liquid carrier, for
example, saline, water-
for-injection, immediately prior to use. Alternatively, the formulations may
be presented for
continuous infusion. Extemporaneous injection solutions and suspensions may be
prepared
from sterile powders, granules and tablets of the kind previously described.
Formulations suitable for rectal administration include suppositories with
standard
carriers such as cocoa butter or polyethylene glycol. Formulations suitable
for topical
administration in the mouth, for example buccally or sublingually, include
lozenges,
containing the active ingredient in a flavored base such as sucrose and acacia
or tragacanth,
and pastilles comprising the active ingredient in a base such as gelatin and
glycerin or sucrose
and acacia. For intra-nasal administration the compounds of the invention may
be used as a
liquid spray, a dispersible powder or in the form of drops. Drops may be
formulated with an
aqueous or non-aqueous base also comprising one or more dispersing agents,
solubilizing
agents and/or suspending agents.
For administration by inhalation the compounds can be conveniently delivered
from
an insufflator, nebulizer, pressurized packs or other convenient means of
delivering an aerosol
spray. Pressurized packs may comprise a suitable propellant such as
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dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane,
carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined
by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compounds
may
take the form of a dry powder composition, for example a powder mix of the
compound and a
suitable powder base such as lactose or starch. The powder composition may be
presented
in unit dosage form, for example, as capsules, cartridges, gelatin or blister
packs from which
the powder may be administered with the aid of an inhalator or insufflators.
Other formulations include implantable devices and adhesive patches; which
release
a therapeutic agent.
When desired, the above described formulations, adapted to give sustained
release of
the active ingredient, may be einployed. The pharmaceutical compositions may
also contain
other active ingredients such as antimicrobial agents, immunosuppressants
and/or
preservatives.
It should be understood that in addition to the ingredients particularly
mentioned
above, the formulations of this invention may include other agents
conventional in the art with
regard to the type of formulation in question. For example, formulations
suitable for oral
administration may include flavoring agents.
It will be apparent to those persons skilled in the art that certain
excipients may be
more preferable depending upon, for instance, the route of administration, the
concentration
of test compound being administered, or whether the treatment uses a
medicament that
includes a protein, a nucleic acid encoding the test compound, or a cell
capable of secreting a
test compound as the active ingredient.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.
Proper formulation is dependent upon the route of administration chosen.
Dosing and Scheduling
Determination of an effective dose range for the medicaments of the present
invention
is well within the capability of those skilled in the art, especially in light
of the detailed
disclosure provided herein. The therapeutically effective dose of a test
compound can be
estimated initially from cell culture assays and/or animal models. For
example, a dose can
be formulated in animal models to achieve a circulating concentration range
that includes the
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IC50 (the dose where 50% of the cells show the desired effects) as determined
in cell culture.
Toxicity and therapeutic efficacy of test compounds also can be determined by
standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically
effective in 50% of the population). The dose ratio between toxic and
therapeutic effects is
the therapeutic index (i. e., the ratio between LD5o and ED50). Compounds
which exhibit
high therapeutic indices are preferable. The data obtained from these cell
culture assays and
animal studies may be used in formulating a dosage range for use in humans.
The dosage of
such compounds may lie within a range of circulating concentrations that
include the ED50
with little or no toxicity. The dosage may vary within this range 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 pl. Dosage amount and interval may be adjusted individually to provide
plasma levels of
the active test compound sufficient to maintain the desired effects.
Especially, for each of the aforementioned conditions, the compositions, e.g.,
polypeptides and organic compounds, can be administered orally or via
injection at a dose
ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult
humans is
generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about
10 g/day, and
most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage
forms of
presentation provided in discrete units may conveniently contain an amount
which is effective
at such dosage or as a multiple of the same, for instance, units containing
about 5 mg to about
500 mg, usually from about 100 mg to about 500 mg.
The dose employed will depend upon a number of factors, including the age and
sex
of the subject, the precise disorder being treated, and its severity. Also the
route of
administration may vary depending upon the condition and its severity. In any
event,
appropriate and optimum dosages may be routinely calculated by those skilled
in the art,
taking into consideration the above-mentioned factors.
Generally, an efficacious or effective amount of one or more REG4 or KIAA0101
inhibitors is determined by first administering a low dose or small amount of
a REG4 and/or
KIAA0101 inhibitor and then incrementally increasing the administered dose or
dosages,
and/or adding a second REG4 and/or KIAA0101 inhibitor as needed, until a
desired effect of
inhibiting or preventing a cancer mediated by aberrant REG4 and/or KIAA0101
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overexpression or intracellular signalling is observed in the treated subject,
with minimal or
no toxic side effects. Applicable methods for determining an appropriate dose
and dosing
schedule for administration of a pharmaceutical composition of the present
invention is
described, for example, in Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 11th Ed., Brunton, et al., Eds., McGraw-Hill (2006), and in
Remington: The
Science and Practice of Pharmacy, 21st Ed., University of the Sciences in
Philadelphia (USIP),
Lippincott Williams & Wilkins (2005), both of which are hereby incorporated
herein by
reference.
Gene Therapy
The siRNA that inhibits expression of REG4 or KIAA0101, the antibody that
neutralizes the activity of REG4, a cell-permeable dominant negative peptides
identified as
inhibits the interaction between KIAA0101lPCNA association may be
therapeutically
delivered using gene therapy to patients suffering from cancers.
Alternatively, a polypeptide
which comprises the neutralizing antibody or QKGIGEFF/SEQ ID NO: 46 of the
present
invention can also be used as the peptides that directly alter the activity of
REG4 or
KIAA0101/PCNA association. In some aspects, gene therapy embodiments include a
nucleic acid sequence encoding a suitable identified peptide of the invention.
In preferred
embodiments, the nucleic acid sequence includes regulatory elements necessary
for
expression of the peptide in a target cell. The nucleic acid may be equipped
to stably insert
into the genome of the target cell (see e.g., Thomas and Capecchi, (1987) Cell
51:503 for a
description of homologous recombination cassettes vectors).
Delivery of the nucleic acids into a patient may be either direct, in which
case the
patient is directly exposed to the nucleic acid or nucleic acid-carrying
vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in vitro, then
transplanted into
the patient. These two approaches are known, respectively, as in vivo or ex
vivo gene
therapy.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
(1993)
Clinical Phartiaacy 12:488-505; Wu and Wu, (1991) Biotherapy 3:87-95;
Tolstoshev, (1993)
Ant2. Rev. Pharrraacol. Toxicol. 33:573-96; Mulligan, (1993) Science 260:926-
32; and Morgan
and Anderson, (1993)Anrz. Rev. Biochem. 62:191-217; (1993) Trends Biotechnol.
11(5):155-
215. Methods commonly known in the art of recombinant DNA teclinology which
can be
used are described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology,
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John Wiley & Sons, NY; and Kriegler, (1990), Gene Transfer and Expression, A
Laboratory
Manual, Stockton Press, NY.
Diagnosing the chemo-radiation therapeutic resistance of a cancer
The term "diagnosing" is intended to encompass predictions and likelihood
analysis.
The present method is intended to be used clinically in making decisions
concerning treatment
modalities, including therapeutic intervention, diagnostic criteria such as
disease stages, and
disease monitoring and surveillance for cancer.
Method for dia ng osing the chemo-radiation therapeutic resistance of a cancer
The expression of the REG4 was found to be specifically elevated in patients
with
the chemo-radiation therapy resistant pancreatic cancer in surgical sample.
Therefore, REG4
gene identified herein as well as its transcription and translation products
find diagnostic
utility as a marker for the chemo-radiation therapeutic resistance of a cancer
and by
measuring the expression of REG4 gene in a cell sample, the chemo-radiation
therapeutic
resistance of a cancer can be diagnosed. Specifically, the present invention
provides a
following method for diagnosing the chemo-radiation therapeutic resistance of
a cancer by
determining the expression level of the REG4 in the subject:
[1] A method for diagnosing the chemo-radiation therapeutic resistance of a
cancer in
a subject, comprising a step of determining the expression level of REG4 gene
in a
subject-derived biological sample, and wherein an increase in the expression
level as
compared to a normal control level of the gene indicates that the subject
suffers from the
cancer of the chemo-radiation therapeutic resistance or is at a risk of the
chemo-
radiation therapeutic resistance.
[2] The method of [1], wherein the expression level is at least 10% greater
than the
normal control level.
[3] The method of [1], wherein the expression level is determined by any of
the
methods selected from the group consisting of:
(a) detecting mRNA of the gene;
(b) detecting a protein encoded by the gene; and
(c) detecting a biological activity of the protein encoded by the gene.
[4] The method of [3], wherein the expression level is determined by detecting
hybridization of a probe to a gene transcript of the gene.
[5] The method of [4], wherein the hybridization step is carried out on a DNA
array.
[6] The method of [3], wherein the expression level is determined by detecting
the
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binding of an antibody against the protein encoded by REG4 gene as the
expression
level of the gene.
[7] The method of [1], wherein the biological sample comprises biopsy, sputum
or
blood.
[S] The method of [1], wherein the subject-derived biological sample comprises
an
epithelial cell.
[9] The metliod of [1], wlierein the subject-derived biological sample
comprises a
cancer cell.
[10] The method of [1], wherein the subject-derived biological sample
comprises a
cancerous epithelial cell.
[11] The method of [1], wherein the cancer is pancreatic cancer.
A subject to be diagnosed by the present method is preferably a mammal.
Exemplary
mammals include, but are not limited to, e.g., human, non-human primate,
mouse, rat, dog,
cat, horse, and cow.
It is preferred to collect a biological sample from the subject to be
diagnosed to
perform the diagnosis. Any biological material can be used as the biological
sample for the
determination so long as it comprises the objective transcription or
translation product of the
REG4. The biological samples include, but are not limited to, bodily tissues
and fluids, such
as blood, sputum and urine. Preferably, the biological sample contains a cell
population
comprising an epithelial cell, more preferably a cancerous epithelial cell or
an epithelial cell
derived from tissue suspected to be cancerous. Further, if necessary, the cell
may be purified
from the obtained bodily tissues and fluids, and then used as the biological
sample.
According to the present invention, the expression level of the REG4 in the
subject-
derived biological sample is determined. The expression level can be
determined at the
transcription (nucleic acid) product level, using methods lcnown in the art.
For example, the
mRNA of the REG4 may be quantified using probes by hybridization methods
(e.g., Northern
hybridization). The detection may be carried out on a chip or an array. The
use of an array
is preferable for detecting the expression level of a plurality of genes
(e.g., various cancer
specific genes) including the REG4. Those skilled in the art can prepare such
probes
utilizing the sequence information of the REG4 (SEQ ID NO: 1 GenBank Accession
No.AY126670). For example, the cDNA of the REG4 may be used as the probes. If
necessary, the probe may be labeled with a suitable label, such as dyes,
fluorescent and
isotopes, and the expression level of the gene may be detected as the
iiitensity of the
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liybridized labels.
Furthermore, the transcription product of the REG4 may be quantified using
primers
by amplification-based detection methods (e.g., RT-PCR). Such primers can also
be
prepared based on the available sequence information of the gene. For example,
the primers
(SEQ ID NO: 3 and 4) used in the Example may be employed for the detection by
RT-PCR or
Northern blot, but the present invention is not restricted thereto.
Specifically, a probe or
primer used for the present method hybridizes under stringent, moderately
stringent, or low
stringent conditions to the mRNA of the REG4. As used herein, the phrase
"stringent
(hybridization) conditions" is indicated in Small interferint! RNA section.
Alternatively, the translation product may be detected for the diagnosis of
the present
invention. For example, the quantity of the REG4 protein may be determined. A
method
for determining the quantity of the protein as the translation product
includes immunoassay
methods that use an antibody specifically recognizing the protein. The
antibody may be
monoclonal or polyclonal. Furthermore, any fragment or modification (e.g.,
chimeric
antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the
detection, so long
as the fragment retains the binding ability to the REG4 protein. Methods to
prepare these
kinds of antibodies for the detection of proteins are well known in the art,
and any method
may be employed in the present invention to prepare such antibodies and
equivalents thereof.
As another method to detect the expression level of the REG4 gene based on its
translation product, the intensity of staining may be observed via
immunohistochemical
analysis using an antibody against the REG4 protein. Namely, the observation
of strong
staining indicates increased presence of the protein and at the same time high
expression level
of the REG4 gene. Furthermore, the translation product may be detected based
on its
biological activity.
The expression level of REG4 gene in a biological sample can be considered to
be
increased if it increases from the control level of REG4 gene by, for example,
10%, 25%, or
50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0
fold, more than 5.0
fold, more than 10.0 fold, or more.
The control level may be determined at the same time with the test biological
sample
by using a sample(s) previously collected and stored from a subject/subjects
whose disease
state (cancerous or non-cancerous) is/are known. Alternatively, the control
level may be
determined by a statistical method based on the results obtained by analyzing
previously
determined expression level(s) of the REG4 gene in samples from subjects whose
disease
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state are known. Furthermore, the control level can be a database of
expression patterns
from previously tested cells. Moreover, according to an aspect of the present
invention, the
expression level of the REG4 gene in a biological sample may be compared to
multiple
control levels, which control levels are determined from multiple reference
samples. It is
preferred to use a control level determined from a reference sample derived
from a tissue type
similar to that of the patient-derived biological sample. Moreover, it is
preferred, to use the
standard value of the expression levels of the REG4 gene in a population with
a known
disease state. The standard value may be obtained by any method known in the
art. For
example, a range of mean 2 S.D. or mean 3 S.D. may be used as standard
value.
In the context of the present invention, a control level determined from a
biological
sample that is known not to be cancerous is called "normal control level". On
the other hand,
if the control level is determined from chemo-radiation therapeutic resistant
biological sample,
it will be called "chemo-radiation therapeutic resistance control level".
When the expression level of the REG4 gene is increased compared to the normal
control level or is similar to the chemo-radiation therapeutic resistance
control level, the
subject may be diagnosed to be suffering from the cancer with the chemo-
radiation
therapeutic resistance or at a risk of the chemo-radiation therapeutic
resistance.
Difference between the expression levels of a test biological sample and the
control
level can be normalized to the expression level of control nucleic acids,
e.g., housekeeping
genes, whose expression levels are known not to differ depending on the
cancerous or non-
cancerous state of the cell. Exemplary control genes include, but are not
limited to, (3-actin,
glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P 1.
Aspects of the present invention are described in the following examples,
which are
not intended to limit the scope of the invention described in the claims.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below.
The invention will be further described in the following examples, which do
not limit
the scope of the invention described in the claims.
Example 1:
1. General Methods
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Clinical samples
Pre-operative and post-operative (3 or 4 weeks after the curative resection)
serum
samples were obtained with informed consent from seven patients who underwent
curative
resection for pancreatic ductal adenocarcinoma. Conventional paraffin-embedded
tissue
sections of PDACs were also obtained from surgical specimens that had been
resected at the
same center. Tissue microarray samples, where 31 PDAC tissues and 2 endocrine-
tumor
tissues were spotted in duplicate, were obtained from ISU ABXIS (Seoul,
Korea).
Cell lines
PDAC cell lines, PK-45P and SUIT-2, were provided by the Cell Resource Center
for Biomedical Research, Tohoku University (Sendai, Japan), and Kyusliu
Medical Center
(Fukuoka, Japan), respectively. MIAPaCa-2 was purchased from American Type
Culture
Collection (ATCC, Rockville, MD). These cell lines were grown in RPMI 1640
(Sigma-
Aldrich, St. Louis, MO) for PK-45P and SUIT-2, Dulbecco's Modified Eagle's
Medium
(Sigma-Aldrich) for MIAPaCa-2 with 10% fetal bovine serum (Cansera
International, Ontario,
Canada) and 1% antibiotic/antimycotic solution (Sigma-Aldrich). Cells were
maintained at
37 C in humidified air with 5% CO2. FreeStyleTM 293 cells (Invitrogen,
Carlsbad, CA) were
suspended in FreeStyleTM 293 Expression Medium (Invitrogen) and were grown in
Erlenmeyer flask (Corning, NY) rotated on an orbital shaker platform at 125
rpm. Cells were
maintained at 37 C in an atmosphere of humidified air with 8% CO2. Pancreatic
cancer cell
lines MIA-PaCa2 and Panc-1, and normal rodent cell line NIH3T3 were purchased
from the
American Type Culture Collection (ATCC, Rockville, MD), which were grown in
Delbecco's
modified Eagle's medium or RPMI1640 (Sigma-Aldrich, St. Louis, MO). Pancreatic
cancer
cell lines PK-59, KLM-1, PK-45P, and PK-1 were provided by the Cell Resource
Center for
Biomedical Research, Tohoku University (Sendai, Japan) and maintained in
RPMI1640
(Sigma-Aldrich); both media were supplemented with 10% fetal bovine serum
(Cansera
International, Ontario, Canada) and 1% antibiotic/antimycotic solution (Sigma-
Aldrich).
Cells were maintained at 37 C in atmospheres of humidified air with 5% CO2.
2. Semi-quantitative RT-PCR for REG4
Purification of PDAC cells, pancreatic cancer cells and normal pancreatic
ductal
epithelial cells were described previously (Nakamura T, et al. Oncogene 2004;
23: 23 85-
400.); RNAs from the purified cell populations and from normal human heart,
lung, liver,
kidney, brain, pancreas (BD Biosciences, Palo Alto, CA) and normal pancreatic
ductal cells
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were subjected to two rounds of amplification by T7-based in vitr-o
transcription (Epicentre
Technologies, Madison, WI) and subsequent synthesis of single-strand cDNA.
Total RNAs
from human pancreatic cancer cell lines were extracted using Trizol reagent
(Invitrogen,
Carlsbad, CA) according to the manufacturer's recommendation. Extracted RNAs
were
treated with DNase I (Roche, Mannheim, Germany) and reverse-transcribed to
single-
stranded cDNAs using oligo (dT) primer with Superscript II reverse
transcriptase (Invitrogen).
The present inventors prepared appropriate dilutions of each single-stranded
cDNA for
subsequent PCR amplification by monitoring a-tubulin (TUBA) as a quantitative
control.
The primer sequences were
5'-AAGGATTATGAGGAGGTTGGTGT-3' (SEQ ID NO: 9) and
5'-CTTGGGTCTGTAACAAAGCATTC-3' (SEQ ID NO: 10) for TUBA;
5'-CCAATTGCTATGGTTACTTCAGG-3' (SEQ ID NO: 3) and
5'-GAAAAACAAGCAGGAGTTGAGTG-3' (SEQ ID NO: 4) for REG4.
5'-AGCTTTGTTGAACAGGCATTT-3' (SEQ ID No.;26) and
5'-GGCAGCAGTACAACAATCTAAGC-3' (SEQ ID No.;27) for KIAA0101
(NM_014736, amino acid sequence set forth in SEQ ID No.;40 encoded by
nucleotide
sequence set forth in SEQ ID No.;39),
5'-CACCCCCACTGAAAAAGAGA-3' (SEQ ID No.;28) and
5'-TACCTGTGGAGCAAGGTGC-3' (SEQ ID No.;29) for,82MG.
All reactions involved initial denaturation at 94 C for 2 minutes followed by
23
cycles (for TUBA) or 28 cycles (for REG4) at 94 C for 30 seconds, 58 C for 30
seconds, and
72 C for 1 minute or 95 C, 5-min initial denaturation step followed by 23
cycles (02MG) or
28 cycles (for KIAA0101) at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s,
on a GeneAmp
PCR system 9700 (PE Applied Biosystems, Foster, CA).
3. Antibody for REG4
The expressing vector of His-tagged full-length human REG4 was transfected
into
293T cells and recombinant REG4 (REG4-His) was purified from its culture media
by the use
of TALON Purification Kit (Clontech, San Diego, CA). The REG4-His protein was
prepared for injection by emulsifying the antigen solution with Freund's
complete adjuvant.
Polyclonal anti-REG4 antibody (pAb) was raised in rabbits (Medical &
Biological
Laboratories, Nagoya, Japan) against the REG4-His protein, and the immune sera
were
purified on affinity columns according to standard protocol. Mouse monoclonal
antibody
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(mAb) was also raised by inoculating REG4-His into BALB/C mice with Freund's
complete
adjuvant. Their lymphocytes were fused with myeloma cell P3U1 and monoclonal
hybrid
cells were generated and validated by the standard technique.
4. Antibodies and recombinant protein for KIAA0101.
The cDNA fragment encoding full-length KIAA0101 (111 amino-acids, NP_055551)
(SEQ
ID No.;15) was generated using KOD-Plus polymerase (TOYOBO) and cloned into
pET21
vector (Novagen). The recombinant KIAA0101 protein fused with polyhistidine
tag at
COOH terminus was expressed in E. coli, BL21 codon plus (Stratagene), and
purified with
Ni-NTA resin (QIAGEN) under native condition according to the supplier's
protocol.
Further purification was performed by use of High Performance Liquid
Chromatography
AKTA explorer (Amersham) equipped with MonoS HR 5/5 (Amersham). The protein
was
immunized into rabbits, and the immune sera were purified on affinity-columns
packed with
Affi-Gel 10 activated affinity media (Bio-Rad) conjugating recombinant
KIAA0101 protein
with accordance of basic methodology. The affinity-purified anti-KIAA0101
polyclonal
antibody was used for detection of KIAA0101 protein.
5. Immunohistochemical staining for REG4.
The sections were deparaffinized and autoclaved for 15 minutes at 108 C in
citrate
buffer, pH 6Ø Endogenous peroxidase activity was quenched by incubation for
30 minutes
in 0.33% hydrogen peroxide diluted in methanol. After incubation with fetal
bovine serum
for blocking, the sections were incubated with anti-REG4 pAb for 1 hour at
room temperature
(1:1000). After washing with PBS, immunodetection was performed with
peroxidase-
labeled anti-mouse immunoglobulin (Envision kit, Dako Cytomation, Carpinteria,
CA).
Finally, the reactants were developed with 3, 3'-diaminobenzidine (Dako) and
the cells were
counter-stained with hematoxylin.
Through genome-wide cDNA microarray analysis, the present inventors identified
dozens of genes that were over-expressed in PDAC cells (Hartupee JC, et al.
Biochim
Biophys Acta 2001; 1518: 287-93.). Among them, the present inventors focused
on REG4
for which the present inventors confirmed its over-expression by RT-PCR in
seven of the nine
microdissected PDAC cell populations examined (Fig. 1A). Its mRNA levels in
PDAC cells
were apparently higher than that of normal pancreas and vital organs including
heart, lung,
kidney, and brain.
Immunohistochemical analysis using pAb to REG4 at another series of PDAC
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tissues revealed strong signals of REG4 Goblet cell-like vesicles (Fig.1B) or
at the cell
surface (Fig.1C) of cancer cells, while acinar cells in normal pancreas showed
faint staining
of REG4 (Fig.1D) and ductal cells and islet cells showed no signal. Adult
vital organs
including heart, lung, kidney, and brain did not show any staining also (data
not shown). In
addition, tissue-microarray with other series of 31 PDAC tissues spotted
showed that 14 of 31
PDACs expressed high levels of REG4, and totally 35 out of 64 PDACs (55%)
showed
positive staining by anti-REG4 antibody. Well-differentiated PDACs (Gl) showed
positive
staining for REG4 more frequently than less differentiated (G2, G3, and G4)
PDACs
(p=0.0001 by x2 test).
6. Establishment of stably REG4-expressing cells.
To create REG4 expression vector, the entire coding sequence of REG4 cDNA
was amplified by PCR using the primer pair with restriction enzyme sites;
5'-CGGAATTCATGGCTTCCAGAAGCATGC-3' (SEQ ID NO.;63) and
5'-ATAAGAATGCGGCCGCTGGTCGGTACTTGCACAGG-3' (SEQ ID
NO.;64)
which contained EcoRl and NotI restriction sites indicated by the first and
second underlines,
respectively. The product was inserted into the EcoRl and Notl sites of
pCAGGSnHC for
expressing a HA-tagged protein. The plasmid was transfected into REG4-negative
PDAC cell
line, PK-45P cells, using FuGENE6 (Roche) according to the manufacture's
recommended
procedures. A population of cells was selected with 0.5 mg/ml Geneticin
(Invitrogen), and
clonal PK-45P cells were sub-cloned by limiting dilution. To assess the levels
of HA-tagged
REG4 expression, several clones were harvested in a lysis buffer containing
50mM Tris-HCI,
pH 7.4, 150mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1mM EDTA, 0.1% protease
inhibitor cocktail III (Calbiochem, San Diego, CA). Samples were centrifuged
and the pellet
was discarded. The amount of protein present in the supernatant was measured
by Bradford
method. Aliquots of 10 g were subjected to 15% SDS-PAGE and detected by
western
blotting using anti-HA antibody (3F10) (Roche). The amount of each protein was
normalized
by and anti-(3-actin antibody (Sigma-Aldrich). Three clones that expressed
REG4
constitutively were established (C 1-6, C2-6, C 10). Control PK-45P cells
transfected with an
empty pCAGGSnHC-HA vector was also established (M1, M3, M6).
7. y-ray and gemcitabine resistance assay.
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3,000 cells of each of REG4-expressing clones (C 1-6, C2-6, C 10) or control
clones
(M1, M3, M6) was seeded into each well of a 96-well culture dish and incubated
in the
medium containing 10% FBS. After 48 hours pre-incubation, cells were y-
irradiated at 1, 5,
10, or 30 Gy using a 60Co source, or treated with 0.1-100,000 nM gemcitabine
for 48 hours.
After 48 hours, viable cells were measured by using Cell-counting kit-8
(DOJINDO). To
evaluate % inhibition value, relative ratio of absorbance-(each
treatment)/absorbance-(no
treatment control) was calculated. For FACS analysis, 48,000 cells/well were
seeded in 6-well
plate, and after 48 hours pre-incubation, cells were y-irradiated at 0, 1, or
5 Gy using a 60Co
source, or treated with lOnM or 50nM gemcitabine for 48 hours. After 48 hours,
cells were
collected, washed with PBS, fixed with cold 70% ethanol, stained with
propidium iodide (10
g/ml) and ribonuclease A (100 g/ml), and subjected to cell cycle analysis
using
FACSCaliburTM Flow Cytometry System (BD) analysis. The percentage of aneuploid
cells
was calculated with cell cycle analysis software (BD CELLQuestTM)
8. Immunohistochemical staining for KIAA0101.
Conventional sections from pancreatic cancer tissues were obtained from
surgical
specimens that were resected under the appropriate informed consent. Sections
from normal
pancreas were purchased from Biochain (Hayward, CA). The sections were
deparaffinized
and autoclaved at 108 C in Dako Cytomation Target Retrieval Solution High pH
(Dako,
Carpinteria, CA) for 15 min. After blocking of endogenous peroxidase and
proteins, the
sections were incubated with anti-KIAA0101 antibody (diluted by 1:200) at room
temperature
for 30 min. After washing with PBS, immunodetection was performed with
peroxidase
labeled anti-rabbit immunoglobulin (Envision kit, Dako). Finally, the
reactants were
developed with 3, 3'-diaminobenzidine (Dako). Counterstaining was performed
using
hematoxylino
9. Northern blot analysis.
The present inventors extracted total RNAs from several pancreatic cancer cell
lines
using TRlzol reagent (Invitrogen, Carlsbad, CA) and performed Northern blot
analysis. After
treatment with DNase I (Nippon Gene, Osaka, Japan), inRNA was purified with
Micro-
FastTrack (Invitrogen), according to the manufacturer's protocols. A 1- g
aliquot of each
mRNA from pancreatic cancer cell lines, as well as those isolated from normal
human adult
heart, lung, liver, ltidney, brain, and pancreas (BD Biosciences, Palo Alto,
CA), were
separated on 1% denaturing agarose gels and transferred onto nylon membranes.
The 702-
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bp probe specific to KIAA0101 was prepared by PCR using the following primer
set: forward
5'- AGCTTTGTTGAACAGGCATTT-3' (SEQ ID No.;1) and reverse
5'-GGCAGCAGTACAACAATCTAAGC-3' (SEQ ID No.;2). flybridization with a
random-primed, a32P-dCTP-labeled probe was carried out according to the
instructions for
Megaprime DNA labeling system (Amersham Biosciences, Buckinghamshire, UK).
Prehybridization, hybridization and washing were performed according to the
supplier's
recommendations. The blots were auto-radiographed with intensifying screens at
-80 C for
days.
10. Small interfering RNA (siRNA)-expressing constructs and transfection for
knock
10 down KIAA0101.
To knock down endogenous KIAA0101 expression in pancreatic cancer cells, the
present inventors used psiU6BX3.0 vector for expression of short hairpin RNA
against a
target gene as described previously (Taniuchi et al., (2005) Cancer Res, 65:
105-12). The
target sequences of the synthetic oligonucleotides for siRNA for KIAA0101 were
as follows:,
#759si; 5'-GCCATATTGTCACTCCTTCTA-3' (SEQ ID No.; 7), and EGFPsi; 5'-
GAAGCAGCACGACTTCTTC-3' (SEQ ID No.; 8) (as a negative control). Pancreatic
cancer
cell lines KLM-1 and MIA-PaCa2, which highly expressed KIAA0101, were plated
onto 10
cm plates, and transfected with 8 g plasmid designed to express siRNA to
KIAA0101 using
FuGENE6 (Roche) according to manufacture's instruction.
Cells were selected by 0.5 mg/ml (for KLM-1) or 0.8 mg/ml (for MIA-PaCa2) of
Geneticin (Sigma-Aldrich) for 5 days, and then harvested to analyze knockdown
effect on
KIAA0101 expression. For colony formation assay, transfectants expressing
siRNAs were
grown for 14 days in media containing Geneticin. After fixation with 4%
paraformaldehyde,
transfected cells were stained with Giemsa solution to assess colony
formation. Cell
viability was quantified using Cell counting kit-8 (DOJTNDO, Kumamoto, Japan).
After 14
days of culture in the Geneticin-containing medium, the solution was added at
a final
concentration of 10%. Following incubation at 37 C for 3 hours, absorbance at
450nm was
measured with a Microplate Reader 550 (Bio-Rad, Hercules, CA).
11. Establishment of exogenous KIAA0101-expressing cells and their growth in
vitro
and in vivo.
KIAA0101 cDNA was prepared by PCR amplification using the forward primer
that included the Kozak sequence and Notl linker, and the reverse primer
including a NotI
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linker. The PCR product was inserted into the Notl sites of the mammalian
expression
vector, pCAGGS/HA for expressing a HA-tagged protein. The pCAGGS-KIAA0101-HA
or
empty pCAGGS/HA mock vector was transfected into PK-45P, which showed faint
expression of KIAA0101, and NIH3T3 cells, which exhibited hardly detectable
expression of
mouse KIAA0101 homologue (NP_080791), by FuGENE6 (Roche) according to the
manufacturer's protocol.
Then, the Geneticin-resistant clones were selected in the culture medium
containing
0.5 mg/ml for PK-45P and 0.9 mg/ml for NIH3T3 of Geneticin. The exogenous
KIAA0101
expression in each clone was confirmed by Western-blot analysis using anti-
FLAG tag
antibody (Sigma-Aldrich) and anti-[i-actin antibody (Sigma-Aldrich). For
growth assay,
7,500 cells of each of KIAA0101 expressing clone (PK45P-KIAA0101) or control
clone
(PK45P-Mock) was seeded into each well of a 24-well culture dish and incubated
in the
medium containing 10% FBS. Cell viability was quantified with MTT assay. The
experiment was repeated at least three times. For in vivo transformation, 5 x
106 cells of one
stable clone NIH3T3-KIAA0101 and one NIH3T3-Mock were inoculated in the right
and left
flank of 8-week nude mice, respectively, and the tumors were harvested after
four weeks.
Each of the tumors was weighted and immunostained by anti-KIAA0101 antibody.
12. Immunoprecipitation and mass-spectrometric analysis for KIAA0101-
associated
complexes.
To isolate proteins that associated with KIAA0101 protein, the present
inventors
performed immunoprecipitation experiments using the anti-KIAA0101 antibody.
Pancreatic
cancer cell lines KLM-1 and PK59, which over-expressed KIAA0101, were lysed in
lysis
buffer (50mM Tris-HCl pH8.0, 150mM NaCI, 0.5% NP-40, Protease Inhibitor
Cocktail Set
III [Calbiochem, San Diego, CA]). Equal amounts of total proteins were
incubated at 40 C
for lh with 2 g of anti-IKIAA0101 polyclonal antibody or a rabbit IgG (Santa
Cruz
Biotechnologies, Santa Cruz, CA). Immunocomplexes were incubated with protein
G
Sepharose (Zymed Laboratories, South San Francisco, CA) for lh and washed with
lysis
buffer.
Co-precipitated proteins were separated in 5-20% gradient SDS-PAGE and stained
3o by silver-staining kits (Wako, Osaka, Japan). Bands that differentiated
proteins precipitated
with anti-KIAA0101 polyclonal antibody from those precipitated with control
IgG were
excised, digested in-gel with trypsin, and analyzed for peptide-mass
fingerprints using an
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AXIMA-CFR MALDI-TOF mass spectrometer (Shimadzu Corporation, Tsukuba, Japan).
Peptide masses were searched with 10-ppm mass accuracy, and protein-database
searches
were performed using the database-fitting program IntelliMarque (Shimadzu).
The protein
binding identified by this strategy was validated by immunoprecipitation using
anti-
KIAA0101, anti-PCNA antibody (Santa-Cruz), anti-POLD1 antibody (Santa-Cruz)
and anti-
FEN1 antibody (Santa-Cruz).
13. Cell-permeable peptide treatment and KiAA0101-PCNA interaction.
To inhibit the interaction between KIAA0101 and PCNA in the dominant-negative
manner, the present inventors designed the PIP box motif peptide of KIAA0101
conjugating
with arginine (R)-repeat cell-permeable peptide (Noguchi et al., (2004) Nat
Med., 10: 305-9).
PIP20 was RRRRRRRRRRRGGG-VRPTPKWf (SEQ ID No.; 9) (PIP
box motif is shown in parentheses, and the conserved residues are shown as
lower case).
PIP20mut replaced the conserved residues in the PIP box motif with alanine:
RRRRRRRRRRRGGG-VRPTPKW{aKGaGEaa)RLSPK (SEQ ID No.; 10). Scramble
peptide was also designed as a negative control; RRRRRRRRRRRGGG-
IFKQWPRGETKPRVLSPKGF (SEQ ID No.; 11). Furthermore, the present inventors also
designed shorter peptide containing the PIP box motif, PIP 16: RRRRRRRRRRRGGG-
TPKW{qKGiGEff)RLSP (SEQ ID No.; 12), PIP16inut; RRRRRRRRRRRGGG-
TPK (SEQ ID No.; 13). They were synthesized by Sigma-Aldrich and
purified by HPLC to more than 95% grade. Cancer cell line KLM-1 with KIAA0101
expression and normal mouse cell line NIH3T3 without expression of mouse
KIAA0101
homologue were treated with serial concentration (5 M, 10pM, and 20 M) of each
of these
cell-permeable peptides. At dayl and day3, the cells were exposed with each
peptide, and at
day5 viable cell number was evaluated by MTT assay, described above.
Example 2
1. siRNA-expressing vector and colony formation assay/MTT assay.
To knock down endogenous REG4 expression in PDAC cells, the present inventors
used psiU6BX3.0 vector for expression of short hairpin RNA against a target
gene as
described previously (Shimokawa T, et al. Cancer Res 2003; 63: 6116-20.). The
target
sequences of the synthetic oligonucleotides for siRNA for REG4 were as
follows: REG4-si2;
5'-GACAGAAGGAAGAAACTCA-3' (SEQ ID NO: 5), and EGFPsi;
5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 6) (as a negative control). PDACs cell
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lines, SUIT-2 (REG4-positive) and MIAPaCa-2 (REG4-negative), were plated onto
6-well
plates, and transfected with plasmid designed to express siRNA (10 g g) using
FuGENE6
(Roche, Basel, Switzerland) according to manufacture's instruction. Plasmids
expressing
siRNAs were prepared by cloning double-stranded oligonucleotides. The
sequences of
paired oligonucleotides are;
5'-CACCGACAGAAGGAAGAAACTCATTCAAGAGATGAGTTTCTTCCTTCTGTC-3'
(SEQ ID NO; 11) and
5'-AAAACTGTCTTCCTTCTTTGAGTAAGTTCTCTACTCAAAGAAGGAAGACAG-3'
(SEQ ID NO; 12) for si2;
Cells were selected by 0.9 mg/ml (for SUIT-2) or 0.8 mg/ml (for MIAPaCa-2) of
Geneticin (Sigma-Aldrich) for 7 days, and then harvested to analyze knockdown
effect on
REG4 expression. For colony formation assay, transfectants expressing siRNAs
were grown
for 7 days in medium containing Geneticin. After fixation with methanol,
transfected cells
were stained with 0.1% of crystal violet solution to assess colony formation.
In MTT assay,
cell viability was quantified using Cell counting kit-8 (DOJINDO, Kumamoto,
Japan). After
7 days of culture in the Geneticin-containing medium, the solution was added
at a final
concentration of 10%. Following incubation at 37 C for 1.5 hours, absorbance
was measured
at 490 nm and at 630 nm as a reference with Microplate Reader 550 (Bio-Rad,
Hercules, CA).
To examine roles of REG4 over-expression in PDAC cell growth, the present
inventors constructed several expression vectors designed to express siRNA
specifically to
REG4 and transfected them into PDAC cell line SUIT-2, which expressed REG4
endogenously at high level. Among the three plasmids the present inventors
tested in SUIT-2
cells, REG4-si2 showed the significant knockdown effect on endogenous REG4
transcript
(Fig.2A), and this transfection resulted in reduction of the viable cells
measured by MTT
assay (Fig.2B) as well as those of the numbers of colonies (Fig.2C), whereas
the transfection
of other plasmids (a negative control of siEGFP) showed no knockdown effect on
REG4
expression and the cell growth of SUIT-2. On the other hand, REG4-si2 did not
affect the
cell viability of MIAPaCa-2, which did not express REG4, excluding a
possibility of the "off-
targeting" effect of REG4-si2 (data not shown). The growth suppressive effect
of this
siRNA-expressing vector (REG4-si2) was correlated well with their gene-
silencing effects,
and these data indicated a critical role of REG4 in pancreatic cancer cell
survival and/or
growth.
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Example 3
1. Generating bio-active recombinant human REG4.
To create the bio-active form of REG4, the entire coding sequence of REG4 cDNA
was amplified by PCR using the primer pair with restriction enzyme sites;
5'-CG{GAATTC}ATGGCTTCCAGAAGCATGC-3' (forward) (SEQ ID NO: 7) and
5'- ATAAGAAT{GCGGCCGC}TGGTCGGTACTTGCACAGG-3' (reverse) (SEQ ID NO:
8) which contained EcoRl and Notl restriction sites shown in parentheses,
respectively. The
product was inserted into the EcoRI and Notl sites of pCAGGS for expressing a
HA-tagged
protein. FreeStyleTM 293 cells were seeded at 1.5 x 105 cells/ml in 30 ml
medium. REG4-
H.A./pCAGGS vectors were transfected with cells using FuGene 6, according to
the instruction
manuals. Culture medium was harvested after 48 hours and recombinant human
REG4
(rhREG4) was purified with HA agarose (Sigma-Aldricli).
2. Immunoprecipitation.
SUIT-2 cells cultured in 10-cm dish were washed and further cultured for 2
days in
serum-free medium. After centrifugation at 10,000xg and 4 C for 15 ininutes,
the
supernatant was treated protein G Sepharose (Zymed Laboratories, San
Francisco, CA) for 1
hour at 4 C. Then pretreated supernatant was added to a mixture of protein G
Sepharose,
which was pre-incubated with anti-REG4 mAb. Incubation was carried out with
gentle
rotation at 4 C for 4 hours followed by two washing steps. Bound proteins were
eluted and
separated on a 15% SDS-PAGE. After electrophoresis separation, proteins were
transferred
to nitrocellulose membranes (Amersham) and probed with anti-REG4 pAb. Protein
bands
were visualized by chemiluminescent detection system (ECL, Amersham).
3. Akt Phospholylation.
To assess the levels of phosphorylated Akt, PK-45P cells were treated with 0,
0.1, 1
or l OnM rhREG4 and with or without 100 g/m1 anti-REG4 mAb for 6 hours.
Following
treatment, the cells were washed with cold PBS and harvested in a lysis buffer
containing 50
mM HEPES, pH 7.5, 200 mM NaCl, 2.5 mM EDTA, 2.5mM EGTA, 10mM NaF, 1mM
Na3VO4, 0.5% Triton X-100, 0.5 mM 1,4-dithiothreitol, 0.1% protease inhibitor
coclctail III
(Calbiochem, San Diego, CA). Samples were centrifuged and the pellet was
discarded.
The amount of protein present in the supernatant was measured by Bradford
method.
Aliquots of 20 g were subjected to 10% SDS-PAGE and detected by western
blotting using
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anti-pSer473 Akt antibody (Abcam, Cambridge, MA). The total amount of Akt
protein was
evaluated by anti-Akt antibody (Santa Cruz Biotechnology, Inc., CA).
To examine an effect of secreted REG4 on pancreatic cancer cell growth, the
present
inventors generated bio-active rhREG4 protein by using mammalian system
(FreeStyleTM
293-F) (Fig.3A), and performed cell growth assay by treating PK-45P cells,
which showed
low expression of REG4, with several concentration (0-lOnM) of rhREG4. Fig.4B
showed
that the presence of REG4 protein in culture medium clearly stimulated cell
proliferation
dose-dependently, which implicated that secreted REG4 functions to promote
cell
proliferation extracellularly and in autocrine/paracrine manner. One of the
downstream
targets of REG family was reported to be Akt signaling pathway (Sekikawa A, et
al.
Gastroenterology 2005; 128: 642-53., Bishnupuri KS, et al. Gastroenterology
2006; 130: 137-
49.). To examine whether our rhREG4 can activate Akt signaling pathway in PDAC
cells,
PK-45P cells were incubated at the presence of serial doses of rhREG4 and the
phosphorylated Akt was detected by Western blot analysis using the antibody
specific to Akt
with 473 serine phosphorylated. The rhREG4 treatment clearly resulted in
increasing
phosphorylation of Akt (Fig.3C), while the total expression level of Akt was
not changed by
the treatment of rhREG4. These data indicated REG4 stimulated cell growth
through Akt
signaling pathway in PDAC cells.
To evaluate the therapeutic potential of anti-REG4 mAb, the present inventors
performed cell growth assay by treating PDAC cells with anti-REG4 mAb. At
first, the
present inventors checked binding affinity of several anti-REG4 mAbs by
immunoprecipitation using cell culture medium. Fig.4A showed that one anti-
REG4 mAb
(34-1) binds endogenous REG4 protein in SUIT-2 culture medium with high
affinity.
Neutralization assay using PK-45P showed that the anti-REG4 mAb clone 34-1
completely
offset the growth-promoting effect by rhREG4 treatment, while the control
antibody did not
show any neutralizing activity (Fig.4B). And the growth assay using SUIT-2
cells
expressing endogenous REG4 at high level showed that anti-REG4 mAb treatment
inhibited
SUIT-2 cell growth dose-dependently (Fig.4C), while anti-REG4 mAb did not
affect the cell
growth of MIAPaCa-2 that did not express REG4 at all.
Furthermore, the present inventors also examined the effect on Akt
phosphorylation
by treating PK45P cell with rhREG4 and anti-REG4 mAb, and anti-REG4 mAb
treatment
suppressed Akt phosphorylation in PDAC cells which was induced by the
treatment of
rhREG4 (Fig.4D), indicating that anti-REG4 mAb was likely to inhibit Alct
signaling
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pathways in PDAC cells by neutralizing secreted REG4 and shutting down its
autocrine/paracrine pathways. Taken together, these data suggested that anti-
REG4 antibody
has neutralization activity to cell proliferation stimulated by REG4.
To evaluate the therapeutic potential of anti-REG4 mAb, the present inventors
performed tumor growth assay by treating PDAC cells inoculated mice with anti-
REG4 mAb.
5x106 cells of REG4-expressing PDAC cell line SUIT2 were inoculated in the
flank of 8-
week nude mice, and the long (L) and short diameters (S) of the tumors were
measured twice
a week. The tumor volumes were calculated by L x L x S x 0.52. The antibody
treatment with
anti-REG4 mouse mAb 34-1 (300 u g/mouse i.p.) started when the tumor volume
reached
100-200 mm3. The antibody was treated two times per a week intraperitoneally,
and as a
control, non-specific mouse IgG (Acris) was also treated at the same schedule.
As shown in
Figure 5A, 34-1 mAb treatment suppressed the tumor growth (P=0.0598) and at
day 30, the
tumors were harvested and weighted. 34-1 mAb treatment suppressed the tumor
weights
significantly (Fig. 513, P=0.0489).
Example 4
1. REG4 expression in chemo-radiation therapy resistant PDAC in vitro.
To investigate how REG4 expression can contribute to the resistance of PDAC to
chemo-radiation therapy, we generated three clones that constitutively
expressed REG4 from
REG4-negative PDAC cells and treated these clones with y-radiation or
gemcitabine in vitro.
Western blot analysis confirmed exogenous REG4 expression in C1-6, C2-6, and
C10 clones,
but not Mock clones (Fig. 6A). Comparing with non-irradiation, y-radiation
suppressed cell
viability of REG4-expressing cells and Mock cells. However, as shown in Fig.
6B, REG4-
expressing cells were less sensitive to y-radiation, and after 30-Gy y-
radiation, viability of
REG4-expressing cells (C1-6, C10, C2-6) were suppressed to 60% comparing with
non-
irradiation, while Mock cell viability were suppressed to less than 40%
(P<0.001). FACS
analysis after y-radiation demonstrated that 1-Gly y-radiation induced 28.7%
of sub-Gl
population (apoptotic cells) of Mock cells, while only 10.73% of REG4-
expressing cells (Fig.
6C). Furthermore, 5-Gly y-radiation induced 46.47 % of sub-G1 population
(apoptotic cells)
of Mock cells, while only 24.68 % of REG4-expressing cells (Fig. 6C). These
finding
implicated that REG4 expression in PK-45P cells could strongly contribute to
the resistance to
y-radiation.
Next, REG4-positive or negative cells were treated with serial concentration
of
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gemcitabine, and evaluated their viability. As shown in Fig. 7A, REG4-
expressing cells (Cl-6,
C10, C2-6) were less sensitive to gemcitabine, comparing with Mock cells. IC50
of
gemcitabine to REG4-expressing cells was about lOOnM at C1-6 and C10, and
about 50nM at
C2-6, which showed less expression of REG4 than C 1-6 and C 10 (Fig. 6A),
while IC50 to
Mock cells was about 30nM. But this difference is not so dominant.
Furthermore, FACS
analysis showed some trend that REG4-expressing cells showed less apoptotic
cells after
50nM gemcitabine treatment, but it is not statistically significant (Fig. 7B).
Treatment of
higher concentration of gemcitabine did not reveal significant difference of
apoptotic cells.
These findings showed REG4 expression in PK-45P cells might have some
contribution to the
resistance to gemcitabine, but this contribution is not so strong. Hence, REG4
expression in
PDAC cells is likely to contribute to its resistance to radiation, rather than
gemcitabine-based
chemotherapy.
2. REG4 expression in the neo-adjuvant chemo-radiation therapy resistant
patient.
In order to evaluate the association between REG4 expression and therapeutic
resistance of pancreatic cancers, the surgical specimens of the patients, who
underwent the
neo-adjuvant chemo-radiation therapy (neo-CRT) followed by surgical resection
of their
pancreatic adenocarcinomas, were investigated. They were treated with 3D
confocal radiation
therapy (total 50 Gy) and, concurrently, gemcitabine (lg/m2/week for 3 weeks),
followed by
8-week follow-up treatment. 19 surgical specimens were immunostained with anti-
REG4
antibody and REG4 expression and the pathological response to neo-CRT were
evaluated.
Among 19 neo-CRT specimens, 9 samples showed histological response to neo-CRT,
while
10 did not. Regarding to REG4 expression, only 2 out of 9 responders showed
REG4
expression in the tumor cells (Fig. 8A and B), while 7 out of 10 non-
responders showed
REG4 expression (Fig. 8C and D). This REG4 expression was significant
associated with the
response to neo-CRT (chi2 test, P=0.0028), implicating that REG4 expression
could make
cancer cells survived under the cytotoxic treatments such as chemotherapy and
radiation
therapy.
Example 5
Over-expression ofKIAA0101 in pancreatic cancer cells.
Among a number of genes that were over-expressed in pancreatic tumor cells on
a
genome-wide cDNA microarray analysis (Nakamura T., Oncogene, 23: 23 85-400,
2004.), the
present inventors focused on KIAA0101 for this study. The present inventor's
microarray
data had shown over-expression of KIAA0101 in all of the informative cases of
pancreatic
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cancer cells the present inventors examined (14 out of 14 informative cases
showed more than
folds of expression signal), and its over-expression was confirmed by RT-PCR
in eight of
the nine microdissected pancreatic cancer cell populations examined (Fig. 9A).
Northern-
blot analysis using a KIAA0101 cDNA fragment as the probe identified a
transcript of about
5 1.2 kb that was highly expressed in all cancer cell lines the present
inventors examined; no
expression was observed in any vital organs including lung, heart, liver and
kidney (Fig. 913).
Immunohistochemical analysis using a polyclonal antibody to KIAA0101 also
showed strong
signals in the nuclei of pancreatic cancer cells in all of the pancreatic
cancer tissue sections
from five additional patients (Fig. 9C). No staining was observed in normal
pancreatic
10 epithelia and acinar cells (Fig. 9C) and in the vital normal organs
including lung, heart, liver
and kidney.
Example 6
Effect of KIAA0101 knockdown by siRNAs on growth of pancreatic cancer cells.
To investigate for the biological significance of ICAA0101 overexpression in
cancer
cells and its potentials as a molecular target for cancer therapy, the present
inventors
constructed several siRNA-expression vectors specific to KIAA0101 mRNA
sequences=and
transfected them into KLM-1 and MIA-PaCa2 that endogenously express high
levels of
IGAA0101 mRNA. A knockdown effect was confirmed by RT-PCR when the present
inventors used #759si constructs (Fig. l0A). Colony-formation assays (Fig.
lOB) and MTT
assays (Fig. lOC) using KLM-1 revealed a drastic reduction in the number of
cells transfected
with #759si, compared with EGFPsi for which no knockdown effect was apparent.
Similar
effects were obtained with the MIA-PaCa2 cell line. These findings indicated
that
KIAA0101 is likely to play some important roles of cancer cell proliferation
and inhibition of
KIAA0101 function can have some potential as a novel molecular target for
cancer therapy.
Example 7
Exogenous overexpression of KiAA0101 promoted cancer cell growth and
transformed
NIH3T3.
To further explore the potential oncogenic property of KIAA0101, the present
inventors established several clones of PK45P-derivative cell lines, PK45P-
1,CIAA0101, in
which exogenous IUAA0101 expressed constitutively. The present inventors also
prepared
control PK-45P cells transfected with the mock vector (PK45P-Mock) and
compared their
growth rates. Western blot analysis (Fig. 11A) validated exogenous KIAA0101
expression
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in six clones. The growth curve measured by MTT assay demonstrated that the
six clones of
PK45P-KIAA0101 (solid lines) grew significantly more rapidly than the four
PK45P-mock
clones (dash lines, Fig. 11B), indicating the KIAA0101 expression enhanced
proliferation of
cancer cells.
Among the cell line the present inventors examined, only normal mouse NIH3T3
cell
line did not showed any expression of KIAA0101 homologue and the present
inventors
enforced KIAA0101 expression in NIH3T3 and investigated whether KIAA0101
overexpression transforms NIH3T3 in vivo. As shown in Fig.11C, tllree clones
of NIH3T3-
KIAA0101 formed the tumors at the right frank of nude mice), while NIH3T3-Mock
did not
at the left frank, and the immunohistochemical staining of these tumors showed
positive
staining of KIAA0101 at the nucleus of tumor cells (Fig. 11D). These results
implicated that
KIAA0101 had its ability to transform normal cells to tumor cells.
Example 8
Identification of PCNA, POLD1, FEN1 as a complex of KIAA0101 protein.
To investigate the biological functions of KIAA0101 further, the present
inventors
carried out iminunoprecipitation to identify complexes with KIAAOlOlprotein,
using a
polyclonal antibody to KIAA0101. Silver-stained immunoprecipitated fractions
separated on
SDS-PAGE gels showed that several proteins were immunoprecipitated with
KIAA0101
proteins from cancer cell lysate, compared with results from a control sample
(Fig. 12A).
Each protein was analyzed by a 1VIALDI-TOF system after in-gel trypsin
digestion; they were
identified as PCNA, POLD1 (polymerase S p125 subunit), and FENl (flap
endonuclease-1).
These interactions were confirmed by immunoprecipitation experiment shown Fig.
12B. All
of these proteins are involved with DNA replication/repair, and POLD1 and FEN1
also bind
to PCNA as well as KIAA0101 (Jonsson et al., (1998) EMBO J. Apr 15;17(8):2412-
25,
Zhang et al., (1999) J Biol Chem. Sep 17;274(38):26647-53; Bruning & Shamoo
(2004)
Structure. Dec;12(12):2209-19.), suggesting that KIAA0101 can involve DNA
replication
through the interaction with PCNA.
Example 9
Inhibition of the interaction between KIAA0101 and PCNA by cell-permeable
dominant-negative peptide.
To inhibit the interaction between KIAA0101 and PCNA through the conserved PIP
box motif of KIAA0101, the present inventors designed the dominant-negative
peptide
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containing this PIP box and conjugated this with arginine (R)-repeat to
facilitate cell
permeability (Fig. 13A). In vitro study, the present inventors validated the
inhibition of the
interaction between PCNA and KIAA0101 by immunoprecipitation. In the presence
of
PIP20, PCNA was not immunoprecipitated with KIAA0101, while PIP20mut where the
conserved residues were replaced with alanine (Fig. 13A) and scramble peptide
did not affect
the interaction (Fig. 13B). Then the present inventors evaluated whether these
peptides
inhibited cancer cell growth by treating cancer cells and normal fibroblast
NIH3T3 cell with
these peptides. Mouse KIAA0101 (NP_080791) has high homology with human one
and
the target region for dominant-negative peptide in human KIAA0101 was 100%
identical with
that in mouse KIAA0101. Fig. 13C demonstrated that PIP20 suppressed cancer
cell growth
dose-dependently, while PIP20mut and scramble peptides did not. On the other
hand, PIP20
did not affect the growth of mouse normal cell line NIH3T3 cells that did not
express the
homologue of human KIAA0101. These findings indicated that PIP20 specifically
inhibited
the cell growth by targeting KIAA0101.
Next the present inventors designed short PIP peptides by deleting some
residues of
N- and C-franking regions with PIP box motif maintained (PIP 16 and PIP 16mut
shown in Fig.
13A) and treated cancer cell line and NIH3T3 cells with them. PIP16 treatment
suppressed
cancer cell growth strongly, however, PIP16 also affected NIH3T3 growth, and
its growth-
suppressive effect was lost by replacing the conserved residues of PIP box
motif with alanines,
as well as PIP20 (Fig. 13D). These findings suggested that the effect of PIP16
was not
specific to KIAA0101 and PIP16 was likely to affect the interaction between
PCNA and other
DNA replication proteins.
Discussion
This invention demonstrated its potentiality as a molecular target for PDAC
treatment. In order to invention the role or function of secreted REG4 in
pancreatic
carcinogenesis or progression, the present inventors lcnocked down the
endogenous REG4
expression by siRNA in PDAC cell lines, and the present inventors exposed
cancer cell with
recombinant REG4. These findings from our experiments indicated that REG4
functions as
an autocrine / paracrine growth factor and mediate Alct signaling pathways via
uiiknown
receptor. But it is unknown how REG4 mediate Akt signaling patliway, and that
is the issue
that should be investigated by further studies, in addition to identification
of REG4 receptor
(Kobayashi S, et al. J Biol Chem 2000; 275: 10723-6.).
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Recent researches reported that REG4 and other family member, REG1, were
likely
to function as anti-apoptotic factor for colon cancer or gastric cancer
through Akt pathway
(Sekikawa A, et al. Gastroenterology 2005; 128: 642-53., Bishnupuri KS, et al.
Gastroenterology 2006; 130: 137-49.), and our study validated this activated
pathways by
treatment of secreted REG4 and Alct signaling pathway is most likely to be the
downstream
pathways of REG family signaling associated with cancer growth or anti-
apoptosis. REG
family is seemed to be expressed in the tissue injury or the regeneration
process and to play
some important roles of tissue regeneration (Unno M, et al. Adv Exp Med Biol
1992; 321: 61-
6., Zhang YW, et al. World J Gastroentero12003; 9: 2635-41.). Considering that
Akt
signaling pathway activation by REG4 and other REG members, they may be
associated with
the sensitivity to chemotherapy or radiation therapy of cancer, and it would
be interesting to
investigate the association between REG4 expression and the effect of chemo-
radiation
therapy in vitro or in vivo.
It is noteworthy that our monoclonal antibody specific to REG4 neutralizes
secreted
REG4 in the culture medium in vitro and treatment of these neutralizing
antibodies
significantly suppressed PDAC cell growth by shutting down REG4
autocrine/paracrine
pathway and blocking the subsequent Akt phosphorylation. These findings
implicate the
feasibility of neutralizing antibody therapy targeting REG4. Bevacizumab, a
humanized
monoclonal antibody to VEGF, is currently approved in combination with
intravenous 5-
fluorouracil-containing regimens for the first-line treatment of metastatic
colorectal cancer.
Besides anti-angiogenesis factor antibody, antibody against circulating
ligands, such as HGF
(Burgess T, et al. Cancer Res. 2006; 66: 1721-9.) and IL-6 (Trikha M, et al.
Clin Cancer Res
2003; 9: 4653-65.), are under review as anti-cancer drugs, and neutralizing-
antibody therapy
targeting REG4 may also provide us with a novel therapeutic strategy for PDACs
and other
cancer expressing REG4.
In conclusion, the present inventors here show the promising feasibility of
REG4 as a
serum diagnostic marker for PDACs and a molecular target for PDAC therapy, and
by
combining a novel strategy targeting REG4 with other screening methods or
other anti-cancer
therapeutic strategies, the prognosis of PDACs will be made more favorable
than the dismal
prognosis at present.
Moreover, the present inventors identified one over-expressing gene, IUAA0101,
in
pancreatic cancer cells. In RNA level, according to the information of the
present inventor's
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microarray analysis on several human cancers and some reports (Peiwen et al.,
(2001)
Oncogene, 20: 484-9; Mizutani et al., (2005) Cancer, 103: 1785-90), many other
cancer cells
also over-expressed KIAA0101 and its expression is observed generally in
highly
proliferating cells. Immunohistochemical analysis using anti-KIAA0101 antibody
showed that
KIAA0101 was highly expressed in cancer cell and in some level at the crypt of
normal
intestinal mucosa and the germinal center of lymph-node where proliferating
cells are present.
And KIAA0101 expression was dependent on cell cycle and its expression was
highest in S
phase, at which DNA replication is most active (data not shown), which
strongly implicates
that KIAA0101 expression is involved with cell proliferation. Indeed knockdown
of
KIAA0101 by siRNA suppressed cell proliferation in cancer cells in this
invention, and
previous report identified KIA0101 as a PCNA-binding protein by yeast two-
hybrid system
(Yu et al., (2001) Oncogene, 20: 484-9).
PCNA is an essential auxiliary protein for the processes of DNA replication
and
DNA repair and acting as a clamp platform that slides along the DNA template,
interacting
with numerous DNA synthesis or metabolic enzymes (Wyman and Botchan, (1995)
Curr
Biol., 5: 334-7; Warbrick, (2000) Bioessays, 22: 997-1006; Krishna et al.,
(1994) Cell, 79:
1233-43). The present inventors here show that K.IAA0101 also binds to PCNA
directly
through its conserved PIP box motif and KIAA0101 is likely to coordinate PCNA
function by
binding with PCNA or competing with other PCNA-binding partners such as p21
(Waga et al.,
(1994) Nature 369: 574-8; Chen et al., (1996) Nucl Acid Res 24: 1727-33;
Kontopidis et al.,
(2005) PNAS, 102: 1871-6). In the present invention, the present inventors
focused on this
PIP box motif and designed the dominant-negative peptide conjugating with cell
permeable
arginine-repeat (Noguchi et al., (2004) Nat Med., 10: 305-9) to inhibit the
interaction between
PCNA and KIAA0101 specifically. PIP20 suppressed cancer cell growth dose-
dependently,
while PIP20mut did not, and PIP20 did not affect the growth ofNIH3T3 which did
not
expressed KIAA0101, suggesting its high specificity.
Interestingly, the present inventors deleted some residues at the franking
regions of
PIP20, maintaining PIP box, to design the shorter peptide PIP 16 and PIP 16,
which more
strongly suppressed cancer cell growth than PIP20, but it seemed to lose its
specificity to
ICAA0101-PCNA interaction. PCNA are interacting with numerous proteins through
their
PIP box motif (Wyman and Botchan, (1995) Curr Biol., 5: 334-7; Warbrick,
(2000) Bioessays,
22: 195-1006; K rishna et al., (1994) Cell, 79: 1233-43; Chen et al., (1996)
Nucl Acid Res
24: 1727-33; Kontopidis et al. (2005) PNAS, 102: 1871-6) and it is possible
that PIP16 can
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inhibit the interaction between PCNA and other many proteins that play
essential roles of cell
proliferation and are ubiquitously expressed. The flanking residues of PIP20
are likely to be
important in specific inhibition between KIAA0101 and PCNA. In the similar
way, the
peptides derived from p21 PIP box, which also can interact with PCNA, are
capable of
arresting and killing cancer cell and inhibiting of PCNA-dependent DNA
replication can be a
promising strategy for cancer strategy (Chen et al., (1996) Nucl Acid Res 24:
1727-33;
Kontopidis et al. (2005) PNAS, 102: 1871-6).
Intracellular protein-protein interactions constitute major control points in
many
signaling pathways, yet have frequently proven a difficult target for small
molecule chemistry,
often reflecting a protein interface that is extensive, shallow, and
llydrophobic (Walenslcy et
al., (2004) Science, 305: 1446-70). Although peptides are attractive
candidates for
stabilizing or disrupting protein-protein interactions, their efficacy as in
vivo reagents is
severely compromised by their loss of secondary structure, susceptibility to
proteolytic
degradation, and difficulty in penetrating intact cells. But a recent report
about the
modification of peptides indicated its feasibility as druggable targets
(Walensky et al., (2004)
Science, 305: 1446-70), and further structural analysis of targeted protein-
protein interaction
(Kontopidis et al., (2005) PNAS, 102: 1871-6) or DDS improvement can provide
the peptide
inhibiting protein-protein interaction with more attraction for drug
development and
promising feasibility for drug development.
Industrial Applicability:
The present inventors have shown that the cell growth is suppressed by small
interfering RNA (siRNA) that specifically target the REG4 gene or KIAA0101
gene. Thus,
this novel siRNAs are useful target for the development of anti-cancer
pharmaceuticals. For
example, agents that block the expression of REG4 or prevent its activity may
find therapeutic
utility as anti-cancer agents, particularly anti-cancer agents for the
treatment of pancreatic
cancer, such as pancreatic ductal adenocarcinoma (PDAC). Similarly, agents
that block the
expression of KIAA0101 or prevent its activity may find therapeutic utility as
anticancer
agents for the treatment of pancreatic cancer, prostatic cancer, breast
cancer, and bladder
cancer.
The present iilventors also have shown that a monoclonal antibody against REG4
neutralized its growth-promoting effects and attenuated significantly the
growth of PDAC
cells. Thus, treatment of disease associated with REG4-expressing cells, for
example,
pancreatic cancer is conveniently carried out using antibodies that bind to
REG4.
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The present inventors also have shown that IKIAA0101 interacts with PCNA, and
the
inhibition of the interaction leads to inhibition of cell proliferation of
cancer cells. Thus,
agents that inhibit the binding between KIAA0101 interacts with PCNA and
prevent its
activity have therapeutic utility as anti-cancer agents.
The present invention thus provides novel polypeptides and other compounds
useful in
treating or preventing cancer. The polypeptides of the present invention are
composed of an
amino acid sequence which contains QKGIGEFF/SEQ ID NO: 46. The polypeptides of
the
present invention can be administered to inhibit the proliferation of, or to
induce apoptosis in,
cancer cells. The polypeptides of the present invention are expected to
exhibit cell
proliferation inhibiting effects against various cancers. Particularly, the
polypeptides of the
present invention have been confirmed to have cell proliferation inhibiting
effects on
pancreatic cancer.
Pancreatic cancer is an important cancer for which an effective treatment
method is
still desired to be provided. Therefore, the present invention is significant
in that it also
provides an effective method for treating and/or preventing pancreatic cancer.
While the invention has been described in detail and with reference to
specific
embodiments thereof, it will be apparent to one skilled in the art that
various changes and
modifications can be made therein without departing from the spirit and scope
of the
invention.
