Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PHARMACEUTICAL PREPARATIONS AND METHODS
FOR INHIBITING TUMORS
FIELD OF THE INVENTION
The present invention relates to pharmaceutical preparations
(i.e., composition) for use as tumor suppressive agents for tumors
arising from cancers such as prostatic adenocarcinoma, stomach
cancer, breast cancer, endometrial and ovarian cancers, and benign
prostate hyperplasia (BPH).
BACKGROUND OF THE INVENTION
The prostate gland, which is found exclusively in male mammals,
produces several components of semen and blood and several regulatory
peptides. The prostate gland comprises stroma and epithelium cells,
the latter group consisting of columnar secretory cells and basal
nonsecretory cells. A proliferation of these basal cells as well as
stroma cells gives rise to benign prostatic hyperplasia (BPH), which
is one common prostate disease. Another common prostate disease is
prostatic adenocarcinoma (CaP), which is the most common of the fatal
pathophysiological prostate cancers, and involves a malignant
transformation of epithelial cells in the peripheral region of the
prostate gland. Prostatic adenocarcinoma and benign prostatic
hyperplasia are two common prostate diseases, which have a high rate
of incidence in the aging human male population. Approximately one
out of every four males above the age of 55 suffers from a prostate
disease of some form or another. Prostate cancer is the second most
common cause of cancer related death in elderly men, with
approximately 96,000 cases diagnosed and about 26,000 deaths reported
annually in the United States.
Studies of the various substances synthesized and secreted by
normal, benign and cancerous prostates carried out in order to gain
an understanding of the pathogenesis of the various prostate diseases
reveal that certain of these substances may be used as
immunohistochemical tumor markers in the diagnosis of prostate
disease. The three predominant proteins or polypeptides secreted by
a normal prostate gland are: (1) Prostatic Acid Phosphatase (PAP);
(2) Prostate Specific Antigen (PSA); and, (3) Prostate Secretory
Protein of 94 amino acids (PSP94), which is also known as Prostatic
Inhibin Peptide (PIP), Human Seminal Plasma Inhibin (HSPI), or -2-
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microseminoprotein (R-MSP), and which is hereinafter referred to as
PSP94.
PSP94 is a simple non-glycosylated cysteine-rich protein, and
constitutes one of three predominant proteins found in human seminal
fluid along with Prostate Specific Antigen (PSA) and Prostate Acid
Phosphatase (PAP). PSP94 has a molecular weight of 10.7 kiloDaltaon
(kDa), and the complete amino acid sequence of this protein has
already been determined (SEQ ID NO:1). The cDNA and gene for PSP94
have been cloned and characterized (Ulvsback, et al., Biochem.
Biophys. Res. Comm., 164:1310, 1989; Green, et al., Biochem. Biophys.
Res. Comm., 167:1184, 1990). Immunochemical and in situ
hybridization techniques have shown that PSP94 is located
predominantly in prostate epithelial cells. It is also present,
however, in a variety of other secretory epithelial cells (Weiber, et
al., Am. J. Pathol., 137:593, 1990). PSP94 has been shown to be
expressed in prostate adenocarcinoma cell line, LNCap (Yang, et al.,
J. Urol., 160:2240, 1998). As well, an inhibitory effect of
exogenous PSP94 on tumor cell growth has been observed both in vivo
and in vitro (Garde, et al., Prostate, 22:225, 1993; Lokeshwar, et
al., Cancer Res., 53:4855, 1993), suggesting that PSP94 could be a
negative regulator for prostate carcinoma growth via interaction with
cognate receptors on tumor cells.
Native PSP94 has been shown to have a therapeutic modality in
treating hormone refractory prostate cancer (and potentially other
prostate indications).
Metabolic and immunohistochemical studies have shown that the
prostate is a major source of PSP94. PSP94 is involved in the
feedback control of, and acts to suppress secretion of, circulating
follicle-stimulating hormone (FSH) both in-vitro and in-vivo in adult
male rats. PSP94 acts both at the pituitary as well as at the
prostate site since both are provided with receptor sites for PSP94.
It has been demonstrated to suppress the biosynthesis and release of
FSH from the rat pituitary as well as to possibly affect the
synthesis/secretion of an FSH-like peptide by the prostate. These
findings suggest that the effects of PSP-94 on tumor growth in vivo,
could be attributed to the reduction in serum FSH levels.
Both PSA and PAP have been studied as tumor markers in the
detection of prostate disease, but since both exhibit elevated levels
in prostates having benign prostatic hyperplasia (BPH), neither
marker is specific and therefore they are of limited utility.
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Recently, it has been shown that PSP94 concentrations in serum
of patients with BPH or CaP are significantly higher than normal.
The highest serum concentration of PSP94 observed in normal men is
approximately 40 ng/ml, while in men with either BPH or CaP, serum
concentrations of PSP94 have been observed in the range from 300-400
ng/ml. Because there exists some overlap in the concentrations of
PSP94 in subjects having normal prostates and patients exhibiting
either BPH or CaP, serum levels in and of themselves are of little
value.
A major therapy in the treatment of prostate cancer is
androgen-ablation. While most patients respond initially to this
treatment, its effectiveness decreases over time, possibly because of
the presence of a heterogenous population of androgen-dependant and
androgen-independent cells to the androgen treatment, while any
androgen insensitive cells present would continue to proliferate
unabated.
Other forms of cancer, which are currently exacting a heavy
toll on population are breast cancer in women and cancer of the
gastrointestinal tract. Currently, the use of various cancer drugs
such as mitomycin, idarubicin, cisplatin, 5-fluoro-uracil,
methotrexate, adriamycin and daunomycin form part of the therapy for
treating such cancers. One drawback to such a therapeutic treatment
is the presence of adverse side effects due to the drugs in the
concentration ranges required for effective treatment.
Accordingly, it would be advantageous to find a more effective
means of arresting the growth of prostate, breast and
gastrointestinal cancer cells and tumors, which may be used
effectively against both androgen sensitive and androgen insensitive
cells.
In previous work, described in United States Patent No.
5,428,011, we provided pharmaceutical preparations (i.e.,
compositions) of native human seminal plasma PSP94 for inhibiting in-
vitro and in-vivo cancerous prostate, gastrointestinal and breast
tumors. The pharmaceutical preparations included native human
seminal plasma PSP94 which could be administered in an appropriate
dosage form, dosage quantity and dosage regimen to a patient
suffering from prostate cancer. In another embodiment, the
pharmaceutical preparation included a mixture of human seminal plasma
PSP94 and an anticancer drug which may be administered in an
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appropriate dosage form, dosage quantity and dosage regimen to a
patient suffering from, for example gastrointestinal cancer.
PSP94 sourced from human seminal fluid carries with it
significant risk of contamination with infectious agents (e.g., HIV,
hepatitis (a, b, or c), and other viruses and/or prions). Even with
the use of harsh chemical treatment, total eradication of such agents
cannot be guaranteed. Additionally, human seminal fluid is found in
limited supply, thus making bulk production of PSP94 very difficult.
Therefore, the acceptability of human or even xenogeneic sourced
PSP94 may be very difficult for both the regulatory authorities and
the marketplace.
Therefore, the use of recombinant technology for producing
PSP94 would represent a significant advancement, as recombinant PSP94
could be produced both free of pathogens and in an unlimited supply.
Furthermore, the material would be homogeneous from a single lot
source, avoiding batch variation.
SUMMARY OF THE INVENTION
In its first aspect the present invention relates to a
polypeptide or a polypeptide analog selected from the group
consisting of the polypeptide as set forth in SEQ ID NO: 3, the
polypeptide as set forth in SEQ ID NO: 4, the polypeptide as set
forth in SEQ ID NO: 5, and the polypeptide as set forth in SEQ ID NO:
6, a polypeptide analog of at least five contiguous amino acids of
SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or
of SEQ ID NO: 6, a polypeptide analog of at least two contiguous
amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ
ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog consisting of the
amino acid sequence X1 W Q X2 D X1 C X1 X2 C X2 C X3 X1 X2 as set forth in
SEQ ID NO: 89, wherein X1 is either glutamic acid (Glu), asparagine
(Asn) or aspartic acid (Asp), X2 is either threonine (Thr) or serine
(Ser), and X3 is either tyrosine (Tyr) or phenylalanine (Phe), a
polypeptide analog comprising SEQ ID NO: 5 and having an addition of
at least one amino acid to its amino-terminus, wherein said
polypeptide analog comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 59 to SEQ ID NO: 88, a polypeptide analog
comprising SEQ ID NO: 5 and having an addition of at least one amino
acid to its carboxy-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group consisting of SEQ
ID NO: 10 to SEQ ID NO: 58, a polypeptide analog comprising two to
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fifty units of SEQ ID NO: 5, a polypeptide analog comprising two to
ten units of SEQ ID NO: 5, a polypeptide analog consisting of a
sequence of from two to fourteen amino acid units wherein the amino
acid units are selected from the group of amino acid units of SEQ ID
NO: 5 consisting of glutamic acid (Glu), tryptophan (Trp), glutamine
(Gln), threonine (Thr), aspartic acid (Asp), asparagine (Asn),
cysteine (Cys), or tyrosine (Tyr), a polypeptide analog having at
least 90 % of its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5, a polypeptide analog having at
least 70 % of its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5, and a polypeptide analog having
at least 50 % of its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5. The polypeptide analog
mentionned herein may be capable of inhibiting the growth of a tumor
or more precisely may be capable of inhibiting the growth of
prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial,
ovarian or other cancers of epithelial secretion, or benign prostate
hyperplasia (BPH).
In a second aspect, the present invention relates to the use of
a polypeptide or a polypeptide analog selected from the group
consisting of rHuPSP94 as set forth in SEQ ID NO: 2, the decapeptide
as set forth in SEQ ID NO: 3, the polypeptide as set forth in SEQ ID
NO: 4 (polypeptide 7-21), the polypeptide as set forth in SEQ ID NO:
5 (PCK3145), and the polypeptide as set forth in SEQ ID NO: 6
(polypeptide 76-94), a polypeptide analog of at least five contiguous
amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ
ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog of at least two
contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID
NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog
consisting of the amino acid sequence X1 W Q X2 D X1 C X1 X2 C X2 C X3 X1
X2 as set forth in SEQ ID NO: 89, wherein X1 is either glutamic acid
(Glu), asparagine (Asn) or aspartic acid (Asp), X2 is either
threonine (Thr) or serine (Ser), and X3 is either tyrosine (Tyr) or
phenylalanine (Phe), a polypeptide analog comprising SEQ ID NO: 5 and
having an addition of at least one amino acid to its amino-terminus,
wherein said polypeptide analog comprising SEQ ID NO:5 is selected
from the group consisting of SEQ ID NO: 59 to SEQ ID NO: 88, a
polypeptide analog comprising SEQ ID NO: 5 and having an addition of
at least one amino acid to its carboxy-terminus, wherein said
polypeptide analog comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a polypeptide analog
comprising two to fifty units of SEQ ID NO: 5, a polypeptide analog
comprising two to ten units of SEQ ID NO: 5, a polypeptide analog
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consisting of a sequence of from two to fourteen amino acid units
wherein the amino acid units are selected from the group of amino
acid units of SEQ ID NO: 5 consisting of glutamic acid (Glu),
tryptophan (Trp), glutamine (Gin), threonine (Thr), aspartic acid
(Asp), asparagine (Asn), cysteine (Cys), or tyrosine (Tyr), a
polypeptide analog having at least 90 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, a
polypeptide analog having at least 70 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, and a
polypeptide analog having at least 50 of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5 and
mixture(s) thereof, for inhibiting the growth of a tumor or more
precisely for inhibiting the growth of prostatic adenocarcinoma,
stomach cancer, breast cancer, endometrial, ovarian or other cancers
of epithelial secretion, or benign prostate hyperplasia (BPH).
In one embodiment of the second aspect of the present
invention, the polypeptide or polypeptide analog may be used with an
anticancer drug, such as, for example, mitomycin, idarubicin,
cisplatin, 5-fluoro-uracil, methotrexate, adriamycin, daunomycin,
taxol (i.e., paclitaxel), taxol derivative (e.g.,docetaxel, taxane),
and mixtures thereof.
In an additional embodiment of the second aspect of the present
invention, the polypeptide or polypeptide analog may be used with a
pharmaceutically acceptable carrier.
In a further embodiment of the second aspect of the present
invention the polypeptide or polypeptide analog may be used with a
time-release means such as, for example, liposomes and
polysaccharides for effecting continual dosing of said polypeptide or
polypeptide analog.
It other embodiments of the second aspect of the present
invention, the polypeptide or polypeptide analog may be used with an
anticancer drug and a pharmaceutically acceptable carrier, with an
anticancer drug and a time-release means, with a pharmaceutically
acceptable carrier and a time-release means, or with an anticancer
drug, a pharmaceutically acceptable and a time-release means. Some
examples of an anticancer drug, a pharmaceutically acceptable carrier
and a time-release means are described herein.
In a third aspect, the present invention relates to a method
for treating a patient with a tumor or more precisely with prostatic
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adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian
or other cancers of epithelial secretion, or benign prostate
hyperplasia (BPH), the method comprising administering to the patient
a pharmaceutical composition comprising a polypeptide or polypeptide
analog selected from the group consisting of rHuPSP94 as set forth in
SEQ ID NO: 2, the decapeptide as set forth in SEQ ID NO: 3, the
polypeptide as set forth in SEQ ID NO: 4 (polypeptide 7-21), the
polypeptide as set forth in SEQ ID NO: 5 (PCK3145), and the
polypeptide as set forth in SEQ ID NO: 6 (polypeptide 76-94), a
polypeptide analog selected from the group consisting of a
polypeptide analog of at least five contiguous amino acids of SEQ ID
NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ
ID NO: 6, a polypeptide analog of at least two contiguous amino acids
of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5,
or of SEQ ID NO: 6, a polypeptide analog consisting of the amino acid
sequence X1 W Q X2 D X, C X1 X2 C X2 C X3 X1 X2 as set forth in SEQ ID NO:
89, wherein X1 is either glutamic acid (Glu), asparagine (Asn) or
aspartic acid (Asp), X2 is either threonine (Thr) or serine (Ser),
and X3 is either tyrosine (Tyr) or phenylalanine (Phe), a polypeptide
analog comprising SEQ ID NO: 5 and having an addition of at least one
amino acid to its amino-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group consisting of SEQ
ID NO: 59 to SEQ ID NO: 88, a polypeptide analog comprising SEQ ID
NO: 5 and having an addition of at least one amino acid to its
carboxy-terminus, wherein said polypeptide analog comprising SEQ ID
NO:5 is selected from the group consisting of SEQ ID NO: 10 to SEQ ID
NO: 58, a polypeptide analog comprising two to fifty units of SEQ ID
NO: 5, a polypeptide analog comprising two to ten units of SEQ ID NO:
5, a polypeptide analog consisting of a sequence of from two to
fourteen amino acid units wherein the amino acid units are selected
from the group of amino acid units of SEQ ID NO: 5 consisting of
glutamic acid (Glu), tryptophan (Trp), glutamine (Gln), threonine
(Thr), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), or
tyrosine (Tyr), a polypeptide analog having at least 90 % of its
amino acid sequence identical to the amino acid sequence set forth in
SEQ ID NO: 5, a polypeptide analog having at least 70 % of its amino
acid sequence identical to the amino acid sequence set forth in SEQ
ID NO: 5, and a polypeptide analog having at least 50 % of its amino
acid sequence identical to the amino acid sequence set forth in SEQ
ID NO: 5 and mixtures thereof. The polypeptide analog mentionned
herein may be capable of inhibiting the growth of a tumor or more
precisely may be capable of inhibiting the growth of prostatic
adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian
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or other cancers of epithelial secretion, or benign prostate
hyperplasia (BPH).
The method for treating a patient as described above may
result, for example, in the inhibition (e.g., reduction, control,
atenuation, prohibition) of the growth of a tumor(s) in a patient
suffering for example from prostatic adenocarcinoma, stomach cancer,
breast cancer, endometrial, ovarian or other cancers of epithelial
secretion, or benign prostate hyperplasia (BPH). The method
described above may be performed, for example, by administering to
the patient a pharmaceutical composition comprising a polypeptide, a
polypeptide analog, or mixtures thereof of the present invention.
In one embodiment of the third aspect of the present invention,
the polypeptide or polypeptide analog may be used with an anticancer
drug, such as, for example, mitomycin, idarubicin, cisplatin, 5-
fluoro-uracil, methotrexate, adriamycin, daunomycin, taxol (i.e.,
paclitaxel), taxol derivative (e.g.,docetaxel, taxane), and mixtures
thereof.
In an additional embodiment of the third aspect of the present
invention, the polypeptide or polypeptide analog may be used with a
pharmaceutically acceptable carrier.
In a further embodiment of the third aspect of the present
invention the polypeptide or polypeptide analog may be used with a
time-release means such as for example, liposomes and polysaccharides
for effecting continual dosing of said polypeptide or polypeptide
analog.
It other embodiments of the third aspect of the present
invention, the polypeptide or polypeptide analog may be used with an
anticancer drug and a pharmaceutically acceptable carrier, with an
anticancer drug and a time-release means, with a pharmaceutically
acceptable carrier and a time-release means, or with an anticancer
drug, a pharmaceutically acceptable and a time-release means. Some
examples of an anticancer drug, a pharmaceutically acceptable carrier
and a time-release means are described herein.
In a fourth aspect, the present invention relates to a method
for treating a patient with a tumor or more precisely with prostatic
adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian
or other cancers of epithelial secretion, or benign prostate
hyperplasia (BPH), the method comprising administering to the patient
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a pharmaceutical composition including a vector comprising the
nucleotide sequence of SEQ ID NO: 9 and a pharmaceutically acceptable
carrier or a pharmaceutical composition comprising a polynucleotide
selected from the group consisting of a polynucleotide having at
least 10 to 285 contiguous residues of SEQ ID NO: 9, and a
polynucleotide having at least 10 to 150 contiguous residues of SEQ ID
NO: 9, and a pharmaceutically acceptable carrier.
In one embodiment of the fourth aspect of the present
invention, the vector or the polynucleotide may be used with an
anticancer drug such as, for example, mitomycin, idarubicin,
cisplatin, 5-fluoro-uracil, methotrexate, adriamycin, daunomycin,
taxol (i.e., paclitaxel), taxol derivative (e.g.,docetaxel, taxane),
and mixtures thereof.
In an additional embodiment of the fourth aspect of the present
invention, the vector or the polynucleotide may be used with a time-
release means such as, for example, liposomes and polysaccharides for
effecting continual dosing of said vector.
In further embodiment of the fourth aspect of the present
invention, the vector or the polynucleotide may be used with an
anticancer drug such as, for example, mitomycin, idarubicin,
cisplatin, 5-fluoro-uracil, methotrexate, adriamycin, daunomycin,
taxol (i.e., paclitaxel), taxol derivative (e.g.,docetaxel, taxane),
and mixtures thereof and with a time-release means such as, for
example, liposomes and polysaccharides for effecting continual dosing
of said vector or polynucleotide.
In a fifth aspect, the present invention relates to a
pharmaceutical composition for inhibiting (e.g., recuding,
controling, atenuating, prohibiting) the growth of a tumor in a
patient suffering from prostatic adenocarcinoma, stomach cancer,
breast cancer, endometrial, ovarian or other cancers of epithelial
secretion, or benign prostate hyperplasia (BPH), comprising:
a)a polypeptide or a polypeptide analog selected from the
group consisting of rHuPSP94 as set forth in SEQ ID NO: 2,
the decapeptide as set forth in SEQ ID NO: 3, the
polypeptide as set forth in SEQ ID NO: 4 (Polypeptide 7-
21), the polypeptide as set forth in SEQ ID NO: 5
(PCK3145), the polypeptide as set forth in SEQ ID NO: 6
(Polypeptide 76-94), a polypeptide analog of at least five
contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3,
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of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a
polypeptide analog of at least two contiguous amino acids
of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ
ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog
consisting of the amino acid sequence X1W Q X D X1 C X1 X2 C
X2 C X3 X1 X: as set forth in SEQ ID NO: 89, wherein X1 is
either glutamic acid (Glu), asparagine (Asn) or aspartic
acid (Asp), X2 is either threonine (Thr) or serine (Ser),
and X3 is either tyrosine (Tyr) or phenylalanine (Phe), a
polypeptide analog comprising SEQ ID NO: 5 and having an
addition of at least one amino acid to its amino-terminus
wherein said polypeptide analog comprising SEQ ID NO:5 is
selected from the group consisting of SEQ ID NO: 59 to SEQ
ID NO: 88, a polypeptide analog comprising SEQ ID NO: 5
and having an addition of at least one amino acid to its
carboxy-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a
polypeptide analog comprising two to fifty units of SEQ ID
NO: 5, a polypeptide analog comprising two to ten units of
SEQ ID NO: 5, a polypeptide analog consisting of a
sequence of from two to fourteen amino acid units wherein
the amino acid units are selected from the group of amino
acid units of SEQ ID NO: 5 consisting of glutamic acid
(Glu), tryptophan (Trp), glutamine (Gln), threonine (Thr),
aspartic acid (Asp), asparagine (Asn), cysteine (Cys), or
tyrosine (Tyr), a polypepti.de analog having at least 90 0
of its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5, a polypeptide analog
having at least '70 % of its amino acid sequence identical
to the amino acid sequence set forth in SEQ ID NO: 5, and
a polypeptide analog having at least 50 % of its amino
acid sequence identical to the amino acid sequence set
forth in SEQ ID NO: 5, and mixture(s) thereof, and;
b)an anticancer drug such as, for example, mitomycin,
idarubicin, cisplatin, 5-fluoro-uracil, methotrexate,
adriamycin, daunomycin, taxol, taxol derivative, and
mixtures thereof.
In one embodiment of the fifth aspect of the present invention
the pharmaceutical composition may further comprise a time-release
means such as, for example, liposomes and polysaccharides for
effecting continual dosing of the composition.
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In a sixth aspect, the present invention relates to a
pharmaceutical composition for inhibiting the growth of a tumor in a
patient suffering from prostatic adenocarcinoma, stomach cancer,
breast cancer, endometrial, ovarian or other cancers of epithelial
secretion, or benign prostate hyperplasia (BPH), comprising:
a) a polypeptide or polypeptide analog selected from the
group consisting of rHuPSP94 as set forth in SEQ ID NO:
2, the decapeptide as set forth in SEQ ID NO: 3, the
polypeptide as set forth in SEQ ID NC): 4 (Polypeptide 7-
21), the polypeptide as set. forth in SEQ ID NO: 5
(PCK3145), the polypeptide as set forth in SEQ ID NO: 6
(Polypeptide 76-94), a polypeptide analog of at least
five contiguous amino acids of SEQ ID NO: 2, of SEQ ID
NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID NO:
6, a polypeptide analog of at least two contiguous amino
acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4,
of SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog
consisting of the amino acid sequence X1 W Q X2 D X1 C X1 X2
C X2 C X3 X1 X2 as set forth in SEQ ID NO: 89, wherein X1 is
either glutamic acid (Glu), asparagine (Asn) or aspartic
acid (Asp), X2 is either threonine (Thr) or serine (Ser),
and X3 is either tyrosine (Tyr) or phenylalanine (Phe), a
polypeptide analog comprising SEQ ID NO: 5 and having an
addition of at least one amino acid to its amino-terminus
wherein said polypeptide analog comprising SEQ ID NO:5 is
selected from the group consisting of SEQ ID NO: 59 to
SEQ ID NO: 88, a polypeptide analog comprising SEQ ID NO:
5 and having an addition of at least one amino acid to
its carboxy-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a
polypeptide analog comprising two to fifty units of SEQ
ID NO: 5, a polypeptide analog comprising two to ten
units of SEQ ID NO: 5, a polypeptide analog consisting of
a sequence of from two to fourteen amino acid units
wherein the amino acid units are selected from the group
of amino acid units of SEQ ID NO: 5 consisting of
glutamic acid (Glu), tryptophan (Trp), glutamine (Gin),
threonine (Thr), aspartic acid (Asp), asparagine (Asn),
cysteine (Cys), or tyrosine (Tyr), a polypeptide analog
having at least 90 % of its amino acid sequence identical
to the amino acid sequence set forth in SEQ ID NO: 5, a
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polypeptide analog having at least 70 % of its amino acid
sequence identical to the amino acid sequence set forth
in SEQ ID NO: 5, and a polypeptide analog having at least
50 % of its amino acid sequence identical to the amino
acid sequence set forth in SEQ ID NO: 5, and mixture(s)
thereof, and;
b) a pharmaceutically acceptable carrier.
In one embodiment of the sixth aspect of the present invention
the pharmaceutical composition may further comprise a time-release
means such as, for example, liposomes and polysaccharides for
effecting continual dosing of the composition.
In a second embodiment of the sixth aspect of the present
invention the pharmaceutical composition may further comprise an
anticancer drug such as, for example, mitomycin, idarubicin,
cisplatin, 5-fluoro-uracil, methotrexate, adriamycin, daunomycin,
taxol, taxol derivative, and mixtures thereof.
In a third embodiment of the sixth aspect of the present
invention, the pharmaceutical composition may further comprise a
time-release means and an anticancer drug. Examples of time-release
means and anticancer drug are described herein.
In a seventh aspect, the present invention relates to a
pharmaceutical composition comprising:
a) A polypeptide or polypeptide analog selected from the
group consisting of rHuPSP94 as set forth in SEQ ID NO: 2,
the decapeptide as set forth in SEQ ID NO: 3, the
polypeptide as set forth in SEQ ID NO: 4 (polypeptide 7-
21), the polypeptide as set. forth in SEQ ID NO: 5
(PCK3145), the polypeptide as set forth in SEQ ID NO: 6
(polypeptide 76-94), a polypeptide analog of at least five
contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3,
of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a
polypeptide analog of at least two contiguous amino acids
of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ
ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog
consisting of the amino acid sequence X1 W Q X2 D X1 C X1 X2 C
X2 C X3 X1 X., as set forth in SEQ ID NO: 89, wherein X1 is
either glutamic acid (Glu), asparagine (Asn) or aspartic
acid (Asp), X2 is either threonine (Thr) or serine (Ser),
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and X3 is either tyrosine (Tyr) or phenylalanine (Phe), a
polypeptide analog comprising SEQ ID NO: 5 and having an
addition of at least one amino acid to its amino-terminus
wherein said polypeptide analog comprising SEQ ID NO:5 is
selected from the group consisting of SEQ ID NO: 59 to SEQ
ID NO: 88, a polypeptide analog comprising SEQ ID NO: 5
and having an addition. of at least one amino acid to its
carboxy-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a
polypeptide analog comprising two to fifty units of SEQ ID
NO: 5, a polypeptide analog comprising two to ten units of
SEQ ID NO: 5, a polypeptide analog consisting of a
sequence of from two to fourteen amino acid units wherein
the amino acid units are selected from the group of amino
acid units of SEQ ID NO: 5 consisting of glutamic acid
(Glu), tryptophan (Trp), glutamine (Gin), threonine (Thr),
aspartic acid (Asp), aspara.gine (Asn), cysteine (Cys), or
tyrosine (Tyr), a polypeptide analog having at least 90 %
of its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5, a polypeptide analog
having at least 70 % of its amino acid sequence identical
to the amino acid sequence set forth in SEQ ID NO: 5, and
a polypeptide analog having at least 50 % of its amino
acid sequence identical to the amino acid sequence set
forth in SEQ ID NO: 5, and mixture(s) thereof, in a
therapeutically effective amount, and;
b) an anticancer drug such as, for example, mitomycin,
idarubicin, cisplatin, 5-fluoro-uracil, methotrexate,
adriamycin, daunomycin, taxol, taxol derivative, and
mixtures thereof in a therapeutically effective amount.
In one embodiment of the seventh aspect of the present
invention the pharmaceutical composition may further comprise a time-
release means such as, for example, liposomes and polysaccharides for
effecting continual dosing of the composition.
In an eighth aspect, the present invention relates to a
pharmaceutical composition comprising:
a)a polypeptide or polypeptide analog selected from the group
consisting of rHuPSP94 as set forth in SEQ ID NO: 2, the
decapeptide as set forth in SEQ ID NO: 3, the polypeptide
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as set forth in SEQ ID NO: 4 (polypeptide 7-21), the
polypeptide as set forth in SEQ ID NO: 5 (PCK3145), the
polypeptide as set forth in SEQ ID NO: 6 (polypeptide 76-
94), a polypeptide analog of at least five contiguous amino
acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of
SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog of
at least two contiguous amino acids of SEQ ID NO: 2, of SEQ
ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID
NO: 6, a polypeptide analog consisting of the amino acid
sequence X1 W Q X2 D X1 C X1 X2 C X2 C X3 X1 X2 as set forth in
SEQ ID NO: 89, wherein X1 is either glutamic acid (Glu),
asparagine (Asn) or aspartic acid (Asp), X2 is either
threonine (Thr) or serine (Ser), and X3 is either tyrosine
(Tyr) or phenylalanine (Phe), a polypeptide analog
comprising SEQ ID NO: 5 and having an addition of at least
one amino acid to its amino-terminus wherein said
polypeptide analog comprising SEQ ID NO:5 is selected from
the group consisting of SEQ ID NO: 59 to SEQ ID NO: 88, a
polypeptide analog comprising SEQ ID NO: 5 and having an
addition of at least one amino acid to its carboxy-
terminus, wherein said polypeptide analog comprising SEQ ID
NO:5 is selected from the group consisting of SEQ ID NO: 10
to SEQ ID NO: 58, a polypeptide analog comprising two to
fifty units of SEQ ID NO: 5, a polypeptide analog
comprising two to ten units of SEQ ID NO: 5, a polypeptide
analog consisting of a sequence of from two to fourteen
amino acid units wherein the amino acid units are selected
from the group of amino acid units of SEQ ID NO: 5
consisting of glutamic acid (Glu), tryptophan (Trp),
glutamine (Gln), threonine (Thr), aspartic acid (Asp),
asparagine (Asn), cysteine (Cys), or tyrosine (Tyr), a
polypeptide analog having at least 90 % of its amino acid
sequence identical to the amino acid sequence set forth in
SEQ ID NO: 5, a polypeptide analog having at least 70 % of
its amino acid sequence identical to the amino acid
sequence set forth in SEQ ID NO: 5, and a polypeptide
analog having at least 50 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID
NO: 5, and mixture(s) thereof, in a therapeutically
effective amount, and;
b) a pharmaceutically acceptable carrier.
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In one embodiment of the eighth aspect of the present invention
the pharmaceutical composition may further comprise a time-release
means such as, for example, liposomes and polysaccharides for
effecting continual dosing of the composition.
In a second embodiment of the eight aspect of the present
invention, the pharmaceutical composition may further comprise an
anticancer drug such as, for example, mitomycin, idarubicin,
cisplatin, 5-fluoro-uracil, methotrexate, adriamycin, daunomycin,
taxol, taxol derivative, and mixtures thereof.
In a third embodiment of the eight aspect of the present
invention, the pharmaceutical composition may further comprise a
time-release means and an anticancer drug. Examples of time-release
means and anticancer drug are described herein.
In a ninth aspect, the present invention relates to a
pharmaceutical composition for inhibiting (reducing, controling,
atenuating, prohibiting) the growth of a tumor in a patient suffering
from prostatic adenocarcinoma, stomach cancer, breast cancer,
endometrial, ovarian or other cancers of epithelial secretion, or
benign prostate hyperplasia (BPH), comprising a vector comprising the
nucleotide sequence of SEQ ID NO: 9 and a pharmaceutically acceptable
carrier, or a polynucleotide selected from the group consisting of a
polynucleotide having at least 10 to 285 contiguous residues of SEQ
ID NO: 9 and a polynucleotide having at least 10 to 50 contiguous
residues of SEQ ID NO: 9, and a pharmaceutically acceptable carrier.
In one embodiment of the ninth aspect of the present invention,
the pharmaceutical composition may further comprise an anticancer
drug such as, for example, mitomycin, idarubicin, cisplatin, 5-
fluoro-uracil, methotrexate, adriamycin, daunomycin, taxol (i.e.,
paclitaxel), taxol derivative (e.g.,docetaxel, taxane), and mixtures
thereof.
In an tenth aspect, the present invention relates to a
pharmaceutical composition for inhibiting the growth of a tumor in a
patient, comprising a vector comprising the nucleotide sequence of
SEQ ID NO: 9 and a pharmaceutically acceptable carrier, or a
polynucleotide selected from the group consisting of a polynucleotide
having at least 10 to 285 contiguous residues of SEQ ID NO: 9 and a
polynucleotide having at least 10 to 50 contiguous residues of SEQ ID
NO: 9, and a pharmaceutically acceptable carrier.
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In one embodiment of the tenth aspect of the present invention,
the pharmaceutical composition may further comprise an anticancer
drug such as, for example, mitomycin, idarubicin, cisplatin, 5-
fluoro-uracil, methotrexate, adriamycin, daunomycin, taxol (i.e.,
paclitaxel), taxol derivative (e.g.,docetaxel, taxane), and mixtures
thereof.
In an eleventh aspect, the present invention relates to a
method for treating patients with a disease characterized by elevated
levels of FSH comprising administering a pharmaceutical composition
in an appropriate dosage form, the pharmaceutical composition
comprising a polypeptide or polypeptide analog selected from the
group consisting of rHuPSP94 as set forth SEQ ID NO: 2, the
decapeptide as set forth in SEQ ID NO: 3, the polypeptide as set
forth in SEQ ID NO: 4, the polypeptide as set forth in SEQ ID NO: 5,
and the polypeptide as set forth in SEQ ID NO: 6, a polypeptide
analog of at least five contiguous amino acids of SEQ ID NO: 2, of
SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a
polypeptide analog of at least two contiguous amino acids of SEQ ID
NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ
ID NO: 6, a polypeptide analog consisting of the amino acid sequence
X1 W Q X, D X1 C X1 X2 C X2 C X3 X1 X, as set forth in SEQ ID NO: 89,
wherein X1 is either glutamic acid (Gl.u), asparagine (Asn) or
aspartic acid (Asp), X, is either threonine (Thr) or serine (Ser),
and X3 is either tyrosine (Tyr) or phenylalanine (Phe) , a polypeptide
analog comprising SEQ ID NO: 5 and having an addition of at least one
amino acid to its amino-terminus, wherein said polypeptide analog
comprising SEQ ID NO:5 is selected from the group consisting of SEQ
ID NO: 59 to SEQ ID NO: 88, a polypeptide analog comprising SEQ ID
NO: 5 and having an addition of at least one amino acid to its
carboxy-terminus, wherein said polypeptide analog comprising SEQ ID
NO:5 is selected from the group consisting of SEQ ID NO: 10 to SEQ ID
NO: 58, a polypeptide analog comprising two to fifty units of SEQ ID
NO: 5, a polypeptide analog comprising two to ten units of SEQ ID NO:
5, a polypeptide analog consisting of a sequence of from two to
fourteen amino acid units wherein the amino acid units are selected
from the group of amino acid units of SEQ ID NO: 5 consisting of
glutamic acid (Glu), tryptophan (Trp), glutamine (Gln), threonine
(Thr), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), or
tyrosine (Tyr), a polypeptide analog having at least 90 % of its
amino acid sequence identical to the amino acid sequence set forth in
SEQ ID NO: 5, a polypeptide analog having at least 70 % of its amino
acid sequence identical to the amino acid sequence set forth in SEQ
ID NO: 5, and a polypeptide analog having at least 50 % of its amino
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acid sequence identical to the amino acid sequence set forth in SEQ
ID NO: 5, and mixtures thereof, and a pharmaceutically acceptable
carrier in a human dose.
In a twelfth aspect, the present invention relates to the use
of a polypeptide or a polypeptide analog selected from the group
consisting of rHuPSP94 as set forth in SEQ ID NO: 2, the decapeptide
as set forth in SEQ ID NO: 3, the polypeptide as set forth in SEQ ID
NO: 4 (polypeptide 7-21), the polypeptide as set forth in SEQ ID NO:
5 (PCK3145), and the polypeptide as set. forth in SEQ ID NO: 6
(polypeptide 76-94), a polypeptide analog of at least five contiguous
amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID NO: 4, of SEQ
ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog of at least two
contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID
NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog
consisting of the amino acid sequence X1 W Q X2 D X1 C X1 X^ C X2 C X3 X1
X2 as set forth in SEQ ID NO: 89, wherein X1 is either glutamic acid
(Glu), asparagine (Asn) or aspartic acid (Asp), X2 is either
threonine (Thr) or serine (Ser), and X3 is either tyrosine (Tyr) or
phenylalanine (Phe), a polypeptide analog comprising SEQ ID NO: 5 and
having an addition of at least one amino acid to its amino-terminus,
wherein said polypeptide analog comprising SEQ ID NO:5 is selected
from the group consisting of SEQ ID NO: 59 to SEQ ID NO: 88, a
polypeptide analog comprising SEQ ID NO: 5 and having an addition of
at least one amino acid to its carboxy-terminus, wherein said
polypeptide analog comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a polypeptide analog
comprising two to fifty units of SEQ ID NO: 5, a polypeptide analog
comprising two to ten units of SEQ ID NO: 5, a polypeptide analog
consisting of a sequence of from two to fourteen amino acid units
wherein the amino acid units are selected from the group of amino
acid units of SEQ ID NO: 5 consisting of glutamic acid (Glu),
tryptophan (Trp), glutamine (Gln), threonine (Thr), aspartic acid
(Asp), asparagine (Asn), cysteine (Cys), or tyrosine (Tyr), a
polypeptide analog having at least 90 1 of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, a
polypeptide analog having at least 70 of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, and a
polypeptide analog having at least 50 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5
and mixture(s) thereof, for treating patients with a disease
characterized by elevated levels of FSH.
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The use of a polypeptide or a polypeptide analog selected from
the group consisting of rHuPSP94 as set forth in SEQ ID NO: 2, the
decapeptide as set forth in SEQ ID NO: 3, the polypeptide as set
forth in SEQ ID NO: 4 (polypeptide 7-21), the polypeptide as set
forth in SEQ ID NO: 5 (PCK3145), the polypeptide as set forth in SEQ
ID NO: 6 (polypeptide 76-94), a polypeptide analog of at least five
contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3, of SEQ ID
NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide analog of
at least two contiguous amino acids of SEQ ID NO: 2, of SEQ ID NO: 3,
of SEQ ID NO: 4, of SEQ ID NO: 5, or of SEQ ID NO: 6, a polypeptide
analog consisting of the amino acid sequence X1 W Q X2 D X1 C X1 X2 C X2
C X3 X1 X2 as set forth in SEQ ID NO: 89, wherein X1 is either glutamic
acid (Glu), asparagine (Asn) or aspartic acid (Asp), X2 is either
threonine (Thr) or serine (Ser), and X3 is either tyrosine (Tyr) or
phenylalanine (Phe), a polypeptide analog comprising SEQ ID NO: 5 and
having an addition of at least one amino acid to its amino-terminus,
wherein said polypeptide analog comprising SEQ ID NO:5 is selected
from the group consisting of SEQ ID NO: 59 to SEQ ID NO: 88, a
polypeptide analog comprising SEQ ID NO: 5 and having an addition of
at least one amino acid to its carboxy-terminus, wherein said
polypeptide analog comprising SEQ ID NO:5 is selected from the group
consisting of SEQ ID NO: 10 to SEQ ID NO: 58, a polypeptide analog
comprising two to fifty units of SEQ ID NO: 5, a polypeptide analog
comprising two to ten units of SEQ ID NO: 5, a polypeptide analog
consisting of a sequence of from two to fourteen amino acid units
wherein the amino acid units are selected from the group of amino
acid units of SEQ ID NO: 5 consisting of glutamic acid (Glu),
tryptophan (Trp), glutamine (Gln), threonine (Thr), aspartic acid
(Asp), asparagine (Asn), cysteine (Cys), or tyrosine (Tyr), a
polypeptide analog having at least 90 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, a
polypeptide analog having at least 70 of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5, and a
polypeptide analog having at least 50 % of its amino acid sequence
identical to the amino acid sequence set forth in SEQ ID NO: 5 and
mixtures thereof for the manufacture of a medicament for the
therapeutic treatment of prostatic adenocarcinoma, stomach cancer,
breast cancer, endometrial, ovarian or other cancers of epithelial
secretion, benign prostate hyperplasia (BPH) or a disease
characterized by elevated levels of FSH.
In accordance with the present invention, rHuPSP94 may be used
in a dosage range from about 10 micrograms/kg/day to about 4
milligrams/kg/day, in a dosage range from about 500 picograms/kg/day
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to about 1 milligram/kg/day, in a dosage range from about 5
nanograms/kg/day to about 10 micrograms/kg/day or in a dosage range
from about 5 nanograms/kg/day to about 500 nanograms/kg/day.
In accordance with the present invention, the decapeptide as
set forth in SEQ ID NO: 3, the polypeptide as set forth in SEQ ID NO:
4, the polypeptide as set forth in SEQ ID NO: 5, the polypeptide as
set forth in SEQ ID NO: 6, and mixtures thereof may be used in a
dosage range from about 100 nanograms/kg/day to about 4
milligrams/kg/day.
In accordance with the present invention, the anticancer drug
may be mixed or not with a polypeptide or polypeptide analog or
mixtures thereof or it may be given separately, by a different route,
or even in a different administration schedule (e.g., a different
time or day).
In accordance with the present invention administration of the
composition may be performed by any suitable routes including
administration by injection via the intra-muscular (IM), subcutaneous
(SC), intra-dermal (ID), intra-venous (IV) or intra-peritoneal (IP)
routes or administration at the mucosal membranes including the oral
and nasal cavity membranes using any suitable means.
In accordance with the present invention, the composition may
be used to treat gastrointestinal cancer.
It is known in the art that the proteins or polypeptides of the
present invention may be made according to methods present in the
art. The polypeptides of the present. invention may be prepared for
example, from bacterial cell extracts, or through the use of
recombinant techniques. Polypeptides of the present invention may,
for example, be produced by transformation (transfection,
transduction, or infection) of a host cell with all or part of a
rHuPSP94 (SEQ ID NO: 2), the decapeptide as set forth in SEQ ID NO:
3, the polypeptide as set forth in SEQ ID NO: 4 (polypeptide 7-21),
the polypeptide as set forth in SEQ ID NO: 5 (PCK3145), and the
polypeptide as set forth in SEQ ID NO: 6 (polypeptide 76-94) encoding
DNA sequence in a suitable expression vehicle. Examples of suitable
expression vehicles comprise for example, plasmids, viral particles,
artificial chromosomes and phages. The entire expression vehicle, or
a part thereof, may be integrated into the host cell genome. In some
circumstances, it is desirable to employ an inducible expression
vector.
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CA 02359650 2001-10-15
Any of a wide variety of expression systems may be used to
provide the recombinant protein. The precise host cell used is not
critical to the invention. Polypeptides of the present invention may
be produced in a prokaryotic host (e.g., E. col.i or B. subtilis) or
in a eukaryotic host (yeast e.g., Saccharomyces or Pichia Pastoris;
mammalian cells, e.g., monkey COS cells, mouse 3T3 cells (Todaro GJ
and Green H., J. Cell Biol. 17: 299-313, 1963), Chinese Hamster Ovary
cells (CHO) (Puck TT et al., J. Exp. Med. 108: 945-956, 1958), BHK,
human kidney 293 cells (ATCC: CRL-1573), or human HeLa cells
(ATCC:CCL-2); or insect cells).
In a yeast cell expression system such as Pichia Pastoris (P.
Pastoris), DNA sequence encoding polypeptides of the present
invention may be cloned into a suitable expression vector such as the
pPIC9 vector (Invitrogen). Upon introduction of a vector containing
the DNA sequence encoding all or part of the polypetides of the
present invention into the P. Pastoris host cells, recombination
event may occur for example in the AOX1 locus. Such recombination
event may place the DNA sequence of the various polypetides of the
present invention under the dependency of the AOX1 gene promoter.
Successful insertion of a gene (DNA sequence) encoding polypeptides
of the present invention may result in an expression of such
polypeptides that is regulated and/or induced by methanol added in
the growth media of the host cell (for reference see Buckholz, R.G.
and Gleeson, M.A.G., Biotechnology, 9:1067-1072,1991; Cregg, J.M., et
al., Biotechnology, 11:905-910, 1993; Sreekrishna, K., et al.,
J.Basic Microbiol., 28:265-278, 1988; Wegner, G.H., FEMS Microbiology
Reviews, 87:279-284, 1990).
In mammalian host cells, a number of viral-based expression
systems may be utilized. For example, in the event where an
adenovirus is used as an expression vector for the polypeptides of
the present invention, nucleic acid sequence may be ligated to an
adenovirus transcription/translation control complex (e.g., the late
promoter and tripartite leader sequence). This chimeric gene may be
inserted into the adenovirus genome, for example, by in vitro or in
vivo recombination. Insertion into a non-essential region of the
viral genome (e.g., region El or E3) may result in a recombinant
virus that is viable and capable of expressing polypeptides of the
present invention in infected hosts.
Proteins and polypeptides of the present invention may also be
produced by plant cells. Expression vectors such as cauliflower
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mosaic virus and tobacco mosaic virus and plasmid expression vectors
(e.g., Ti plasmid) may be used for the expression of polypeptides in
plant cells. Such cells are available from a wide range of sources
(e.g., the American Type Culture Collection, Rockland, Md.). The
methods of transformation or transfection and the choice of
expression vehicle are of course to be chosen accordingly to the host
cell selected.
In an insect cell expression system such as Autographa
californica nuclear polyhedrosis virus (AcNPV), which grows in
Spodoptera frugiperda cells, AcNPV may be used as a vector to express
foreign genes. For example, DNA sequence coding for all or part of
the polypeptides of the present invention may be cloned into non-
essential regions of the virus (for example the polyhedrin gene) and
placed under control of an AcNPV promoter, (e.g., the polyhedrin
promoter). Successful insertion of a gene (i.e.,DNA sequence)
encoding polypeptides of the present invention may result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat encoded
by the polyhedrin gene). These recombinant viruses may be used to
infect spodoptera frugiperda cells in which the inserted gene is
expressed.
In addition, a host cell may be chosen for its ability to
modulate the expression of the inserted sequences, or to modify or
process the gene product in a specific, desired fashion. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the protein.
Different host cells have characteristics and specific mechanisms for
posttranslational processing and modification of proteins and gene
products. Of course, cell lines or host systems may be chosen to
ensure desired modification and processing of the foreign protein
expressed. To this end, eukaryotic host cells that possess the
cellular machinery for proper processing of the primary transcript,
glycosylation, and phosphorylation of the gene product may be used.
Such mammalian host cells comprise for example, but are not limited
to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, and 3T3.
Alternatively, polypeptides of the present invention may be
produced by a stably transfected mammalian cell line. A number of
vectors suitable for stable transfection of mammalian cells are
available to the public; methods for constructing such cell lines are
also publicly available. In one example, cDNA encoding the rHuPSP94
protein may be cloned into an expression vector that includes the
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CA 02359650 2009-11-12
dihydrofolate reductase (DHFR) gene. Integration of the plasmid and,
therefore, DNA sequence of polypeptides of the present invention,
into the host cell chromosome may be selected for by including
methotrexate in the cell culture media. This selection may be
accomplished in most cell types.
Specific initiation signals may also be required for the
efficient translation of DNA sequences inserted in a suitable
expression vehicle as described above. These signals may include the
ATG initiation codon and adjacent sequences. For example, in the
event where gene or cDNA encoding polypeptides of the present
invention, would not have their own initiation codon and adjacent
sequences, additional translational control signals may be needed.
For example, exogenous translational control signals, including,
perhaps, the ATG initiation codon, may be needed. It is known in the
art that the initiation codon must be in phase with the reading frame
of the polypeptide sequence to ensure proper translation of the
desired polypeptide. Exogenous translational control signals and
initiation codons may be of a variety of origins, including both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer elements,
transcription terminators.
As may be appreciated, a number of modifications may be made to
the polypeptides and fragments of the present invention without
deleteriously affecting the biological activity of the polypeptides
or fragments. Polypeptides of the present invention comprises for
example, those containing amino acid sequences modified either by
natural processes, such as posttranslational processing, or by
chemical modification techniques which are known in the art.
Modifications may occur anywhere in a polypeptide including the
polypeptide backbone, the amino acid side-chains and the amino or
carboxy termini. It will be appreciated that the same type of
modification may be present in the same or varying degrees at several
sites in a given polypeptide. Also, a given polypeptide may contain
many types of modifications. Polypeptides may be branched as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched and branched cyclic polypeptides may
result from posttranslational natural processes or may be made by
synthetic methods. Modifications comprise for example, without
limitation, acetylation, acylation, addition of acetamidomethvl (Acm)
group, ADP-ribosylation, amidation, covalent attachment to fiavin,
covalent attachment to a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
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or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation and
ubiquitination (for reference see, Protein-structure and molecular
proterties, 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New-
York, 1993).
Other type of polypeptide modification may comprises for
example, amino acid insertion (i.e., addition), deletion and
substitution (i.e., replacement), either conservative or non-
conservative (e.g., D-amino acids, desamino acids) in the polypeptide
sequence where such changes do not substantially alter the overall
biological activity of the polypeptide. Polypeptides of the present
invention comprise for example, biologically active mutants,
variants, fragments, chimeras, and analogs; fragments encompass amino
acid sequences having truncations of one or more amino acids, wherein
the truncation may originate from the amino terminus (N-terminus),
carboxy terminus (C-terminus), or from the interior of the protein.
Analogs of the invention involve an insertion or a substitution of
one or more amino acids. Variants, mutants, fragments, chimeras and
analogs may have the biological property of polypeptides of the
present invention which is to inhibit growth of prostatic
adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian
or other cancers of epithelial secretion, or benign prostate
hyperplasia (BPH).
Example of substitutions may be those, which are conservative
(i.e., wherein a residue is replaced by another of the same general
type). As is understood, naturally occurring amino acids may be sub-
classified as acidic, basic, neutral and polar, or neutral and non-
polar. Furthermore, three of the encoded amino acids are aromatic.
It may be of use that encoded polypept_i.des differing from the
determined polypeptide of the present invention contain substituted
codons for amino acids, which are from the same group as that of the
amino acid be replaced. Thus, in some cases, the basic amino acids
Lys, Arg and His may be interchangeable; the acidic amino acids Asp
and Glu may be interchangeable; the neutral polar amino acids Ser,
Thr, Cys, Gln, and Asn may be interchangeable; the non-polar
aliphatic amino acids Gly, Ala, Val, Ile, and Leu are interchangeable
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CA 02359650 2001-10-15
but because of size Gly and Ala are more closely related and Val, Ile
and Leu are more closely related to each other, and the aromatic
amino acids She, Trp and Tyr may be interchangeable.
It should be further noted that if the polypeptides are made
synthetically, substitutions by amino acids, which are not naturally
encoded by DNA may also be made. For example, alternative residues
include the omega amino acids of the formula NH2(CH2)nCOOH wherein n
is 2-6. These are neutral nonpolar amino acids, as are sarcosine, t-
butyl alanine, t-butyl glycine, N-methyl isoleuci_ne, and norleucine.
Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and
methionine sulfoxide are neutral nonpolar, cysteic acid is acidic,
and ornithine is basic. Proline may be substituted with
hydroxyproline and retain the conformation conferring properties.
It is known in the art that mutants or variants may be
generated by substitutional mutagenesis and retain the biological
activity of the polypeptides of the present invention. These
variants have at least one amino acid residue in the protein molecule
removed and a different residue inserted in its place. For example,
one site of interest for substitutional mutagenesis may include but
are not restricted to sites identified as the active site(s), or
immunological site(s). Other sites of interest may be those, for
example, in which particular residues obtained from various species
are identical. These positions may be important for biological
activity. Examples of substitutions identified as "conservative
substitutions" are shown in table 1. If such substitutions result in
a change not desired, then other type of substitutions, denominated
"exemplary substitutions" in table 1, or as further described herein
in reference to amino acid classes, are introduced and the products
screened.
In some cases it may be of interest to modify the biological
activity of a polypeptide by amino acid substitution, insertion, or
deletion. For example, modification of a polypeptide may result in
an increase in the polypeptide's biological activity, may modulate
its toxicity, may result in changes in bioavailability or in
stability, or may modulate its immunological activity or
immunological identity. Substantial modifications in function or
immunological identity are accomplished by selecting substitutions
that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation. (b)
the charge or hydrophobicity of the molecule at the target site, or
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(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side chain properties:
(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),
Valine (Val), Leucine (Leu), Isoleucine (Ile)
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine
(Thr)
(3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)
(4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),
Lysine (Lys), Arginine (Arg)
(5) residues that influence chain orientation: Glycine (Gly),
Proline (Pro); and
(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine
(Phe)
Non-conservative substitutions will entail exchanging a member
of one of these classes for another.
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TABLE 1. Preferred amino acid substitution
Original residue Exemplary substitution Conservative
substitution
Ala (A) Val, Leu, Ile Val
Arg (R) Lys, Gln, Asn Lys
Asn (N) Gln, His, Lys, Arg Gln
Asp (D) Glu Glu
Cys (C) Ser Ser
Gln (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Asn, Gln, Lys, Arg Arg
Ile (I) Levi, Val, Met, Ala, Leu
Phe, norleucine
Leu (L) Norleucine, Ile, Val, Ile
Met, Ala, Phe
Lys (K) Arq, Gln, Asn Arg
Met (M) Leu, Phe, Ile Leu
Phe (F) Leu, Val, Ile, Ala Leu
Pro (P) Gly Gly
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr Tyr
Tyr (Y) Trp, Phe, Thr, Ser Phe
Val (V) Ile, Leu, Met, Phe, Leu
Ala, norleucine
Example of analogs of PCK3145 (SEQ ID NC): 5) exemplified by
amino acid substitutions has been illustrated below.
Position 1 5 10 15
PCK3145 E W Q T D N C E T C T C Y E T
SEQ ID NO: 89 X, W Q X2 D X, C X, X2 C X, C X3 X, X2
For example, X1 could be glutamic acid (i.e., glutamate) (Glu),
aspartic acid (aspartate) (Asp), or asparagine (Asn), X2 could be
threonine (Thr) or serine (Ser) and X_; could be tyrosine (Tyr) or
phenylalanine (Phe).
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Amino acids sequence insertions (e.g., additions) include amino
and/or carboxyl-terminal fusions ranging in length from one residues
to polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Other insertional variants include the fusion of the N- or C-terminus
of the protein to a homologous or heterologous polypeptide forming a
chimera. Chimeric polypeptides (i.e., chimeras, polypeptide analog)
comprise sequence of the polypeptides of the present invention fused
to homologous or heterologous sequence. Said homologous or
heterologous sequence encompass those which, when formed into a
chimera with the polypeptides of the present invention retain one or
more biological or immunological properties. Examples of homologous
sequences fused to PCK3145 (SEQ ID NO: 5) are illustrated below (1 to
79). Such homologous sequences are derived as it is the case for
PCK3145, from rHuPSP94 (SEQ ID NO: 2).
1) EWQTDNCETCTCYETE (SEQ ID NO: 10)
2) EWQTDNCETCTCYETEI (SEQ ID NO: 11)
3) EWQTDNCETCTCYETEIS (SEQ ID NO: 12)
4) EWQTDNCETCTCYETEISC (SEQ ID NO: 13)
5) EWQTDNCETCTCYETEISCC (SEQ ID NO: 14)
6) EWQTDNCETCTCYETEISCCT (SEQ ID NO: 15)
7) EWQTDNCETCTCYETEISCCTL (SEQ ID NO: 16)
8) EWQTDNCETCTCYETEISCCTLV (SEQ ID NO: 17)
9) EWQTDNCETCTCYETEISCCTLVS (SEQ ID NO: 18)
10) EWQTDNCETCTCYETEISCCTLVST (SEQ ID NO: 19)
11) EWQTDNCETCTCYETEISCCTLVSTP (SEQ ID NO: 20)
12) EWQTDNCETCTCYETEISCCTLVSTPV (SEQ ID NO: 21)
13) EWQTDNCETCTCYETEISCCTLVSTPVG (SEQ ID NO: 22)
14) EWQTDNCETCTCYETEISCCTLVSTPVGY (SEQ ID NO: 23)
15) EWQTDNCETCTCYETEISCCTLVSTPVGYD (SEQ ID NO: 24)
16) EWQTDNCETCTCYETEISCCTLVSTPVGYDK (SEQ ID NO: 25)
17) EWQTDNCETCTCYETEISCCTLVSTPVGYDKD (SEQ ID NO: 26)
18) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDN (SEQ ID NO: 27)
19) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNC (SEQ ID NO: 28)
20) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQ (SEQ ID NO: 29)
21) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQR (SEQ ID NO: 30)
22) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRI (SEQ ID NO: 31)
23) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIF (SEQ ID NO: 32)
24) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFK (SEQ ID NO: 33)
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25) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKK (SEQ ID NO: 34)
26) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKE (SEQ ID NO: 35)
27) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKED (SEQ ID NO: 36)
28) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDC (SEQ ID NO: 37)
29) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCK (SEQ ID NO: 38)
30) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKY (SEQ ID NO: 39)
31) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYI (SEQ ID NO: 40)
32) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIV
(SEQ ID NO: 41)
33) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVV
(SEQ ID NO: 42)
34) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVE
(SEQ ID NO: 43)
35) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEK
(SEQ ID NO: 44)
36) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKK
(SEQ ID NO: 45)
37) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKD
(SEQ ID NO: 46)
38) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDP
(SEQ ID NO: 47)
39) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPK
(SEQ ID NO: 48)
40) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKK
(SEQ ID NO: 49)
41) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
(SEQ ID NO: 50)
42) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKTC
(SEQ ID NO: 51)
43) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKTCS
(SEQ ID NO: 52)
44) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSV (SEQ ID NO: 53)
45) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSVS (SEQ ID NO: 54)
46) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSVSE (SEQ ID NO: 55)
47) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSVSEW (SEQ ID NO: 56)
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CA 02359650 2001-10-15
48) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSVSEWI (SEQ ID NO: 57)
49) EWQTDNCETCTCYETEISCCTLVSTPVGYDKDNCQRIFKKEDCKYIVVEKKDPKKT
CSVSEWII (SEQ ID NO: 58)
50) SCYFIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 88)
51) CYFIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 87)
52) YFIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 86)
53) FIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 85)
54) IPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 84)
55) PNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 83)
56) NEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 82)
57) EGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 81)
58) GVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 80)
59) VPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 79)
60) PGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 78)
61) GDSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 77)
62) DSTRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 76)
63) STRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 75)
64) TRKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 74)
65) RKCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 73)
66) KCMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 72)
67) CMDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 71)
68) MDLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 70)
69) DLKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 69)
70) LKGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 68)
71) KGNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 67)
72) GNKHPINSEWQTDNCETCTCYET (SEQ ID NO: 66)
73) NKHPINSEWQTDNCETCTCYET (SEQ ID NO: 65)
74) KHPINSEWQTDNCETCTCYET (SEQ ID NO: 64)
75) HPINSEWQTDNCETCTCYET (SEQ ID NO: 63)
76) PINSEWQTDNCETCTCYET (SEQ ID NO: 62)
77) INSEWQTDNCETCTCYET (SEQ ID NO: 61)
78) NSEWQTDNCETCTCYET (SEQ ID NO: 60)
79) SEWQTDNCETCTCYET (SEQ ID NO: 59)
Other type of chimera generated by homologous fusion includes
new polypeptides formed by the repetition of two or more polypeptides
of the present invention. The number of repeat may be, for example,
between 2 and 50 units (i.e., repeats). In some instance, it may be
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useful to have a new polypeptide with a number of repeat greater than
50. Examples of new polypeptides formed by the repetition of PCK3145
(SEQ ID NO: 5) are illustrated below (80 to 82) . In some instance,
SEQ ID NO: 5 units may be separated by a linker or an adaptor of
variable length.
80) EWQTDNCETCTCYETEEWQTDNCETCTCYETE (SEQ ID NO: 90)
81) EWQTDNCETCTCYETEEWQTDNCETCTCYETEEWQTDNCETCTCYETE (SEQ ID
NO: 91)
82) EWQTDNCETCTCYETEEWQTDNCETCTCYETEEWQTDNCETCTCYETEEWQTDNCE
TCTCYETE (SEQ ID NO: 92)
Heterologous fusion includes new polypeptides made by the
fusion of polypeptides of the present invention with heterologous
polypeptides. Such polypeptides may include but are not limited to
bacterial polypeptides (e.g., betalactamase, glutathione-S-
transferase, or an enzyme encoded by the E.coli trp locus), yeast
protein, viral proteins, phage proteins, bovine serum albumin,
chemotactic polypeptides, immunoglobulin constant region (or other
immunoglobulin regions), albumin, or ferritin.
Other type of polypeptide modification includes amino acids
sequence deletions (e.g., truncations). Those generally range from
about 1 to 30 residues, more preferably about 1 to 10 residues and
typically about 1 to 5 residues.
A host cell transformed or transfected with nucleic acids
encoding the polypeptides of the present invention (i.e., vector
containing the DNA sequence of the polypeptides of the present
invention) or chimeric proteins formed with the polypeptides of the
present invention are also encompassed by the invention. Any host
cell, which produces a polypeptide analog, mutant, variant, fragment,
or chimera having at least one of the biological properties of the
present invention is encompassed by the present invention. For
example, such host cell may include bacterial, yeast, plant, insect
or mammalian cells. In addition, the polypeptides of the present
invention may be produced in transgenic animals. Transformed or
transfected host cells and transgenic animals may be obtained using
materials and methods that are routinely available to one skilled in
the art.
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DEFINITIONS
General Molecular Biology
Unless otherwise indicated, the recombinant DNA techniques utilized
in the present invention are standard procedures, known to those skilled in
the art. Example of such techniques are explained in the literature in
sources such as J. Perbal, A Practical Guide to Molecular Cloning, John
Wiley and Sons (1984), J. Sambrook et al ., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor),
Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL
Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A
Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.
Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene
Pub. Associates and Wiley-Interscience (1988, including all updates until
present).
"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified
RNA or DNA. "Polynucleotides" include, without limitation single- and
double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single- and double-stranded RNA, and RNA that is a mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded or a
mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to triple-stranded regions comprising RNA or DNA or
both RNA and DNA. The term polynucleotide also includes DNAs or RNAs
containing one or more modified bases and DNAs or RNAs with backbones
modified for stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications has been made to DNA and RNA; thus "polynucleotide" embraces
chemically, enzymatically or metabolically modified forms of
polynucleotides as typically found in nature, as well as the chemical forms
of DNA and RNA characteristic of viruses and cells. "Polynucleotide"
includes but is not limited to linear and end-closed molecules.
"Polynucleotide" also embraces relatively short polynucleotides, often
referred to as oligonucleotides.
"Polypeptides" refers to any peptide or protein comprising two or more
amino acids joined to each other by peptide bonds or modified peptide bonds
(i.e., peptide isosteres). "Polypeptide" refers to both short chains,
commonly referred as peptides, oligopeptides or
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CA 02359650 2001-10-15
oligomers, and to longer chains generally referred to as proteins.
As described above, polypeptides may contain amino acids other than
the 20 gene-encoded amino acids.
As used herein the term "polypeptide analog" relates to
mutants, variants, chimeras, fusions, deletions, additions and any
other type of modifications made relative to a given polypeptide.
As used herein, the term "homologous" sequence relates to
nucleotide or amino acid sequence derived from the rHuPSP94 DNA
sequence or polypeptide.
As used herein, the term "heterologous" sequence relates to DNA
sequence or amino acid sequence of a heterologous polypeptide and
includes sequence other than that of PSP94.
As used herein, the term "tumor" relates to solid or non-solid
tumors, metastasic or non-metastasic tumors, tumors of different
tissue origin including, but not limited to, tumors originating in
the liver, lung, brain, lymph node, bone marrow, adrenal gland,
breast, colon, pancreas, prostate, stomach, or reproductive tract
(cervix, ovaries, endometrium etc.). The term "tumor" as used herein,
refers also to all neoplastic cell growth and proliferation, whether
malignant or benign, and all pre-cancerous and cancerous cells and
tissues.
As used herein, the term "polysaccharide" refers to a substance
made of two or more saccharide unit and comprise, for example,
chitosan, pectin, chondroitin sulfate, cyclodextrin, dextrans, guar
gum, inulin, amylose, and locust bean gum.
As used herein, the term "vector" refers to an autonomously
replicating DNA or RNA molecule into which foreign DNA or RNA
fragments are inserted and then propagated in a host cell for either
expression or amplification of the foreign DNA or RNA molecule. The
term << vector >> comprises and is not limited to a plasmid (e.g.,
linearized or not) that can be used to transfer DNA sequences from
one organism to another.
As used herein, the term "time-release encapsulation means"
refers to controlled or sustained release obtained when a
pharmaceutical composition is formulated, for example, with
polysaccharides, biocompatible polymers, other polymeric matrices,
capsules, microcapsules, microparticles, bolus preparations, osmotic
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pumps, diffusion devices, liposomes, lipospheres, dry powders, or
transdermal delivery systems. Other controlled release compositions of the
present invention include liquids that, upon administration to a mammal,
form a solid or a gel in situ. Furthermore, the term "time-release
encapsulation means" or "time-release means" comprises a class of
biodegradable polymers useful in achieving controlled release of a drug,
for example, polylactic acid, polyglycolic acid, copolymers of polylactic
and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and
crosslinked or amphipathic block copolymers of hydrogels.
As used herein, "pharmaceutical composition" means therapeutically
effective amounts of the agent together with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or
carriers. A "therapeutically effective amount" as used herein refers to
that amount which provides a therapeutic effect for a given condition and
administration regimen. Such compositions are liquids or lyophilized or
otherwise dried formulations and include diluents of various buffer content
(e.g., Tris-HC1., acetate, phosphate), pH and ionic strength, additives
such as albumin or gelatin to prevent absorption to surfaces, detergents
(e.g., TweenTM 20, TweenTM 80, PluronicTM F68, bile acid salts). Solubilizing
agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,
ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal,
benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g.,
lactose, mannitol), covalent attachment of polymers such as polyethylene
glycol to the protein, complexation with metal ions, or incorporation of
the material into or onto particulate preparations of polymeric compounds
such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto
liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts, or spheroplasts. Such compositions will influence the
physical state, solubility, stability, rate of in vivo release, and rate of
in vivo clearance. Controlled or sustained release compositions include
formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also
comprehended by the invention are particulate compositions coated with
polymers (e.g., poloxamers or poloxamines). Other embodiments of the
compositions of the invention incorporate particulate forms protective
coatings, protease inhibitors or permeation enhancers for various routes of
administration, including parenteral, pulmonary, nasal and oral routes. In
one embodiment the pharmaceutical composition is administered parenterally,
paracancerally, transmucosally,
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transdermally, intramuscularly, intravenously, intradermally,
subcutaneously, intraperitonealy, intraventricularly, intracranially
and intratumorally.
Further, as used herein "pharmaceutically acceptable carrier"
or "pharmaceutical carrier" are known in the art and include, but are
not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or
0.8 % saline. Additionally, such pharmaceutically acceptable carriers
may be aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's orfixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers such as those
based on Ringer's dextrose, and the like. Preservatives and other
additives may also be present, such as, for example, antimicrobials,
antioxidants, collating agents, inert gases and the like.
Mutants, variants and analogs proteins
Mutant polypeptides will possess one or more mutations, which
are deletions (e.g., truncations), insertions (e.g., additions), or
substitutions of amino acid residues. Mutants can be either
naturally occurring (that is to say, purified or isolated from a
natural source) or synthetic (for example, by performing site-
directed mutagenesis on the encoding DNA or made by other synthetic
methods such as chemical synthesis) . It is thus apparent that the
polypeptides of the invention can be either naturally occurring or
recombinant (that is to say prepared from the recombinant DNA
techniques).
A protein at least 50 % identical, as determined by methods
known to those skilled in the art (for example, the methods described
by Smith, T.F. and Waterman M.S. (1981) Ad. Appl.Math., 2:482-489, or
Needleman, S.B. and Wunsch, C.D. (1970) J.Mol.Biol., 48: 443-453), to
those polypeptides of the present invention are included in the
invention, as are proteins at least 70 % or 80 % and more preferably
at least 90 % identical to the protein of the present invention.
This will generally be over a region of at least 5, preferably at
least 20 contiguous amino acids.
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"Variant" as the term used herein, is a polynucleotide or
polypeptide that differs from reference polynucleotide or polypeptide
respectively, but retains essential properties. A typical variant of
a polynucleotide differs in nucleotide sequence from another,
reference polynucleotide. Changes in the nucleotide sequence of the
variant may or may not alter the amino acid sequence of a polypeptide
encoded by the reference polynucleotide. Nucleotide changes may
result in amino acid substitutions, additions, deletions, fusion and
truncations in the polypeptide encoded by the reference sequence, as
discussed herein. A typical variant of a polypeptide differs in
amino acid sequence from another, reference polypeptide. Generally,
differences are limited so that the sequence of the reference
polypeptide and the variant are closely similar overall and, in many
regions, identical. A variant and reference polypeptide may differ
in amino acid by one or more substitutions, additions, deletions, or
any combination therefore. A substituted or inserted amino acid
residue may or may not be one encoded by the genetic code. A variant
polynuclotide or polypeptide may be a naturally occurring such as an
allelic variant, or it may be a variant that is riot known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques or by direct
synthesis.
Amino acid sequence variants may be prepared by introducing
appropriate nucleotide changes into DNA, or by in vitro synthesis of
the desired polypeptide. Such variant include, for example,
deletions, insertions, or substitutions of residues within the amino
acid sequence. A combination of deletion, insertion and substitution
can be made to arrive at the final construct, provided that the final
protein product possesses the desired characteristics. The amino
acid changes also may alter posttranslational processes such as
changing the number or position of the glycocylation sites, altering
the membrane anchoring characteristics, altering the intra-cellular
location by inserting, deleting or otherwise affecting the
transmembrane sequence of the native protein, or modifying its
susceptibility to proteolytic cleavage.
It is to be understood herein, that if a "range" or "group" of
substances (e.g. amino acids), substitutents" or the like is
mentioned or if other types of a particular characteristic (e.g.
temperature, pressure, chemical structure, time, etc.) is mentioned,
the present invention relates to and explicitly incorporates herein
each and every specific member and combination of sub-ranges or sub-
groups therein whatsoever. Thus, any specified range or group is to
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CA 02359650 2001-10-15
be understood as a shorthand way of referring to each and every
member of a range or group individually as well as each and every
possible sub-ranges or sub-groups encompassed therein; and similarly
with respect to any sub-ranges or sub-groups therein. Thus, for
example,
- with respect to a pressure greater than atmospheric, this is
to be understood as specifically incorporating herein each
and every individual pressure state, as well as sub-range,
above atmospheric, such as for example 2 psig, 5 psig, 20
psig, 35.5 psig, 5 to 8 psig, 5 to 35, psig 10 to 25 psig,
to 40 psig, 35 to 50 psig, 2 to 100 psig, etc..;
- with respect to a temperature greater than 100 C, this is
15 to be understood as specifically incorporating herein each
and every individual temperature state, as well as sub-
range, above 100 C, such as for example 101 C, 105 C and
up, 110 C and up, 115 C and up, 110 to 135 C, 115 c to
135 C, 102 C to 150 C, up to 210 C, etc.;
- with respect to a temperature lower than 100 C, this is to
be understood as specifically incorporating herein each and
every individual temperature state, as well as sub-range,
below 100 C, such as for example 15 C and up, 15 C to 40
C, 65 C to 95 C, 95 C and lower, etc.;
- with respect to residence or reaction time, a time of 1
minute or more is to be understood as specifically
incorporating herein each and every individual time, as well
as sub-range, above 1 minute, such as for example 1 minute,
3 to 15 minutes, 1 minute to 20 hours, I to 3 hours, 16
hours, 3 hours to 20 hours etc.;
- with respect to polypeptides, a polypeptide analog
consisting of at least two contiguous amino acids of a
particular sequence is to be understood as specifically
incorporating each and every individual possibility, such as
for example, a polypeptide analog consisting of amino acid 1
and 2, a polypeptide analog consisting of amino acids 2 and
3, a polypeptide analog consisting of amino acids 3 and 4, a
polypeptide analog consisting of amino acids 6 and 7, a
polypeptide analog consisting of amino acids 9 and 10, a
polypeptide analog consisting of amino acids 36 and 37, a
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polypeptide analog consisting of amino acids 93 and 94, etc.
with respect to polypeptides, a polypeptide analog
consisting of at least five contiguous amino acids of a
particular sequence is to be understood as specifically
incorporating each and every individual possibility, such as
for example, a polypeptide analog consisting of amino acids
1 to 5, a polypeptide analog consisting of amino acids 2 to
6, a polypeptide analog consisting of amino acids 3 to 7, a
polypeptide analog consisting of amino acids 6 to 10, a
polypeptide analog consisting of amino acids 9 to 13, a
polypeptide analog consisting of amino acids 36 to 40, a
polypeptide analog consisting of amino acids 90 to 94, etc.
- with respect to polypeptides, a polypeptide analog
comprising a particular sequence and having an addition of
at least one amino acid to its amino-terminus is to be
understood as specifically incorporating each and every
individual possibility, such as for example, a polypeptide
analog having an addition of one amino acid to its amino-
terminus, a polypeptide analog having an addition of two
amino acid to its amino-terminus, a polypeptide analog
having an addition of three amino acid to its amino-
terminus, a polypeptide analog having an addition of ten
amino acid to its amino-terminus, a polypeptide analog
having an addition of eighteen amino acid to its amino-
terminus, a polypeptide analog having an addition of forty
amino acid to its amino-terminus, a polypeptide analog
having an addition of two hundred amino acid to its amino-
terminus, etc.
with respect to polypeptides, a polypeptide analog
comprising a particular sequence and having an addition of
at least one amino acid to its carboxy-terminus is to be
understood as specifically incorporating each and every
individual possibility, such as for example, a polypeptide
analog having an addition of one amino acid to its carboxy-
terminus, a polypeptide analog having an addition of two
amino acid to its carboxy-terminus, a polypeptide analog
having an addition of five amino acid to its carboxy-
terminus, a polypeptide analog having an addition of twenty
amino acid to its carboxy-terminus, a polypeptide analog
having an addition of fifty-three amino acid to its carboxy-
terminus, a polypeptide analog having an addition of three
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CA 02359650 2001-10-15
hundred amino acid to its carboxy-terminus, etc.
with respect to polypeptides, a polypeptide analog
comprising two to fifty units of a particular sequence is to
be understood as specifically incorporating each and every
individual possibility, such as for example, a polypeptide
analog comprising two units of that particular sequence, a
polypeptide analog comprising three units of that particular
sequence, a polypeptide analog comprising six units of that
particular sequence, a polypeptide analog comprising
thirteen units of that particular sequence, a polypeptide
analog comprising thirty-five units of that particular
sequence, a polypeptide analog comprising fifty units of
that particular sequence, etc.
- with respect to polypeptides, a polypeptide analog
comprising two to ten units of a particular sequence is to
be understood as specifically incorporating each and every
individual possibility, such as for example, a polypeptide
analog comprising two units of that particular sequence, a
polypeptide analog comprising three units of that particular
sequence, a polypeptide analog comprising four units of that
particular sequence, a polypeptide analog comprising five
units of that particular sequence, a polypeptide analog
comprising six units of that particular sequence, a
polypeptide analog comprising seven units of that particular
sequence, a polypeptide analog comprising eight units of
that particular sequence, a polypeptide analog comprising
nine units of that particular sequence, and a polypeptide
analog comprising ten units of that particular sequence.
- with respect to polypeptides, a polypeptide analog
consisting of a sequence of from two to fourteen amino acid
units wherein the amino acid units are selected from the
group of amino acid units of SEQ ID NO: 5 consisting of
glutamic acid (Glu), tryptophan (Trp), glutamine (Gin),
threonine (Thr), aspartic acid (Asp), asparagine (Asn),
cysteine (Cys), or tyrosine (Tyr), is to be understood as
specifically incorporating each and every individual
possibility, such as for example, a polypeptide analog of
two amino acid units wherein the amino acids are
sequentially; Glu and Trp, a polypeptide analog of two amino
acid units wherein the amino acids are sequentially; Trp and
Glu, a polypeptide analog of three amino acid units wherein
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CA 02359650 2001-10-15
the amino acids are sequentially; Trp, Glu, Trp, a
polypeptide analog of three amino acid units wherein the
amino acids are sequentially; Trp, Trp, Trp, a polypeptide
analog of three amino acid units wherein the amino acids are
sequentially; Glu, Glu, Trp, a polypeptide analog of three
amino acid units wherein the amino acids are, independently
of the order; Tyr, Asp, Glu, a polypeptide analog of three
amino acid units wherein the amino acids are, independently
of the order; Thr, Asp, Asn, a polypeptide analog of three
amino acid units wherein the amino acids are, independently
of the order; Thr, Thr, Asn, a polypeptide analog of four
amino acid units wherein the amino acids are, independently
of the order; Glu, Gln, Cys, Asn, a polypeptide analog of
four amino acid units wherein the amino acids are,
independently of the order; Gln, Gln Cys, Trp, a polypeptide
analog of four amino acid units wherein the amino acids are,
Cys, Cys, Cys, Cys, a polypeptide analog of fourteen amino
acid units wherein the amino acids are, independently of the
order; Asn, Asp, Glu, Gin, Trp, Cys, Tyr, Thr, Thr, Asp,
Asn, Gln, Thr, Cys, a polypeptide analog of fourteen amino
acid units wherein the amino acids are, independently of the
order; Asp, Asp, Asp, Asp, Trp, Cys, Cys, Trp, Thr, Thr,
Thr, Thr, Thr, Cys, a polypeptide analog of fourteen amino
acid units wherein the amino acids are, independently of the
order; Tyr, Tyr, Tyr, Tyr, Tyr, Tyr, Tyr, Tyr, Tyr, Tyr,
Tyr, Tyr, Tyr, Tyr, etc.
- with respect to polypeptides, a polypeptide analog having at
least 90 % of its amino acid sequence identical to a
particular amino acid sequence is to be understood as
specifically incorporating each and every individual
possibility (excluding 100 %), such as for example, a
polypeptide analog having 90 of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 91 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 93 of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 97 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 99 % of its amino acid sequence
identical to that particular amino acid sequence, etc.
- with respect to polypeptides, a polypeptide analog having at
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least 70 % of its amino acid sequence identical to a
particular amino acid sequence is to be understood as
specifically incorporating each and every individual
possibility (excluding 100 %), such as for example, a
polypeptide analog having 70 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 71 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 73 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 88 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 97 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 99 % of its amino acid sequence
identical to that particular amino acid sequence, etc.
with respect to polypeptides, a polypeptide analog having at
least 50 % of its amino acid sequence identical to a
particular amino acid sequence is to be understood as
specifically incorporating each and every individual
possibility (excluding 100 %), such as for example, a
polypeptide analog having 50 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 51 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 54 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 66 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 70 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 79 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 82 % of its amino acid sequence
identical to that particular amino acid sequence, a
polypeptide analog having 99 % of its amino acid sequence
identical to that particular amino acid sequence, etc.
and similarly with respect to other parameters such as low
pressures, concentrations, elements, etc...
It is also to be understood herein that "g" or "gm" is a
reference to the gram weight unit; that "C" is a reference to the
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CA 02359650 2001-10-15
Celsius temperature unit; and "psig" is a reference to "pounds per
square inch gauge".
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts mass spectrometry analysis of polypeptide 7-21 (SEQ
ID NO: 4).
Figure 2 depicts mass spectrometry analysis of polypeptide PCK3145
(SEQ ID NO: 5).
Figure 3 depicts mass spectrometry analysis of polypeptide 76-94 (SEQ
ID NO: 6).
Figure 4a is a graph depicting the in-vitro inhibitory activity of
the decapeptide of SEQ ID NO: 3 on PC-3 cells after 9 days of
culture.
Figure 4b is a graph depicting the in-vitro inhibitory activity of
the native PS294 (nPSP94) on PC-3 cells after 9 days of culture.
Figure 5a is a graph depicting the in-vitro inhibitory activity of
the decapeptide of SEQ ID NO: 3 on PC-3 cells after 21 days of
culture.
Figure 5b is a graph depicting the in-vitro inhibitory activity of
the native PSP94 (nPSP94) on PC-3 cells after 21 days of culture.
Figure 6a is a graph depicting the in-vitro inhibitory activity of
the decapeptide of SEQ ID NO: 3 on PC-3 cells after 10 days of
culture.
Figure 6b is a graph depicting the in-vitro inhibitory activity of
the native PS294 (nPSP94) on PC-3 cells after 10 days of culture.
Figure 7 depicts a gel showing DNA fragmentation following treatment
of PC-3 cells with polypeptide PCK3145 as set forth in SEQ ID NO: 5.
Figure 8 is a graph depicting the results of an apoptosis assay with
an ELISA plus kit following polypeptide treatment of PC-3 cells for
72 hours with various concentration of polypeptide 7-21 (SEQ ID NO:
4), polypeptide PCK3145 (SEQ ID NO: 5), polypeptide 76-94 (SEQ ID NO:
6) or native PSP94 (SEQ ID NO: 1).
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Figure 9 is a graph depicting in vitro fibroblast, cell growth when
exposed for 72 hours to various concentration of native PSP94
(nPSP94) (SEQ ID NO: 1) or various concentration of rHuPSP94 (SEQ ID
NO: 2) or polypeptide 7-21 (SEQ ID NO: 4), polypeptide PCK3145 (SEQ
ID NO: 5), or polypeptide 76-94 (SEQ ID NO: 6).
Figure 10 is a graph depicting the effect of polypeptide 7-21 (SEQ ID
NO: 4), polypeptide PCK3145 (SEQ ID NO: 5), polypeptide 76-94 (SEQ ID
NO: 6), and polypeptide 61-75 on the in vitro growth of PC-3 cells
after 72 hours.
Figure 11 is a graph depicting the effect of polypeptide 22-36 and
polypeptide PCK3145 (SEQ ID NO: 5) on in vitro growth of PC-3 cells
after 72 hours.
Figure 12 is a graph depicting results of study no. MLL-1 on the
anti-tumor efficacy validation of rHuPSP94 (rPSP94) (SEQ ID NO: 2)
against Mat Ly Lu (MLL) tumor implanted in nude mice.
Figure 13 is a graph depicting results of study no. MLL-2 on the
anti-tumor efficacy validation of rHuPSP94 (rPSP94) (SEQ ID NO: 2)
against Mat Ly Lu (MLL) tumor implanted in nude mice.
Figure 14 is a graph depicting tumor volume (tumor growth reduction)
in rHuPSP94-treated nude mice.
Figure 15 is a graph depicting tumor volume (tumor growth reduction)
in decapeptide (SEQ ID NO: 3)-treated nude mice.
Figure 16 is a graph depicting tumor volume (tumor growth reduction)
in control scrambled polypeptide (PB111)-treated mice.
Figure 17 is a graph depicting tumor volume (tumor growth reduction)
in native-PSP94 (nPSP94)-treated mice.
Figure 18 is a graph depicting the in vitro inhibitory activity of
PCK3145 (SEQ ID NO: 5) on PC-3 cells, after a 72 hours treatment, as
measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)
assay.
Figure 19 is a graph depicting the in vitro inhibitory activity of
native PSP94 (SEQ ID NO: 1) and PCK3145 (SEQ ID NO: 5) (GMP grade) on
PC-3 cells, after 48 hours of treatment, as measured by MTS assay.
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Figure 20 is a graph depicting the in vitro inhibitory activity of
PCK3145 (SEQ ID NO: 5) (GMP grade) on PC-3 cells (ATCC), after 72
hours of treatment, as measured by the MTS assay.
Figure 21 is a graph depicting the in vitro inhibitory activity of
PCK3145 (SEQ ID NO: 5)(GMP grade) on PC-3 cells (ATCC), after a 48 or
72 hours treatment, as measured by the MTS assay.
Figure 22 is a graph depicting the in vitro inhibitory activity of
decapeptide as set forth in SEQ ID NO: 3, polypeptide 7-21 as set
forth in SEQ ID NO: 4, polypeptide PCK3145 as set forth in SEQ ID NO:
5, or polypeptide 76-94 as set forth in SEQ ID NO: 6 on PC-3 cells,
measured by [3H]-Thymidine uptake assay.
Figure 23 is a graph depicting the in vitro inhibitory activity of
decapeptide as set forth in SEQ ID NO: 3, polypeptide 7-21 as set
forth in SEQ ID NO: 4, polypeptide PCK3145 as set forth in SEQ ID NO:
5, or polypeptide 76-94 as set forth in SEQ ID NO: 6 on PC-3 cells,
measured by [3H]-Thymidine uptake assay.
Figure 24 is a graph depicting the in vitro inhibitory activity of
native PSP94 (SEQ ID NO: 1) on PC-3 cells after 72 hours treatment,
measured by [3H]-Thymidine uptake assay.
Figure 25 depicts a gel showing DNA fragmentation following treatment
of PC-3 cells with PCK3145 (SEQ ID NO: 5) or doxorubicin.
Figure 26 is a graph depicting the in vivo inhibitory activity of
PCK3145 (SEQ ID NO: 5) (0.1 g/kg/day and 10 g/kg/day) against human
PC-3 tumor xenografted in nude mice.
Figure 27 is a graph depicting the in vivo inhibitory activity of
PCK3145 (SEQ ID NO: 5) (10 g/kg/day to 1000 pg/kg/day, administered
either via the intra-venous or intra-peritoneal route) against human
PC-3 tumor xenografted in nude mice.
Figure 28 is a graph depicting the in vivo inhibitory activity of
polypeptide 7-21 (SEQ ID NO: 4), PCK3145 (SEQ ID NO: 5) or
polypeptide 76-94 (SEQ ID NO: 6), given at doses of 1 pg/kg/day or 10
g/kg/day, in Copenhagen rats implanted with Dunning Mat Ly Lu
tumors.
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Figure 29 is a graph depicting the in vivo inhibitory activity of
PCK3145 (SEQ ID NO: 5) or the scrambled polypeptide given at doses of
pg/kg/day or 100 g/kg/day, in Copenhagen rats implanted with
Dunning Mat Ly Lu tumors.
5
Figure 30 is a graph depicting tumor weight at day 18 following
PCK3145 (SEQ ID NO: 5) or scrambled polypeptide treatment (10
g/kg/day or 100 g/kg/day), in Copenhagen rats implanted with
Dunning Mat Ly Lu tumors.
Figure 31 is a graph depicting the efficacy of PCK3145 and taxotere
(i.e. docetaxel) combination treatment in Nude mice implanted with
PC-3 tumor cells in tumor growth retardation.
DETAILED DESCRIPTION OF THE INVENTION
The recombinant human rHuPSP94 expressed in yeast is non-
glycosylated and has 10 cystein residues. The molecular weight of
rHuPSP94 was determined to be 11.5 kDa, compared to 10.7 kDa for its
native counterpart.
Various experimental studies have been carried out in order to
determine the efficacy of rHuPSP94 (SEQ ID NO: 2) relative to the
native PSP94 secreted by the diseased prostate as tumor suppressive
agent. Studies have also been carried out to determine the efficacy
of the decapeptide as set forth in SEQ ID NO: 3, the polypeptide as
set forth in SEQ ID NO: 4 (polypeptide 7-21), the polypeptide as set
forth in SEQ ID NO: 5 (PCK3145), and the polypeptide as set forth in
SEQ ID NO: 6 (polypeptide 76-94), as tumor suppressive agents. The
tumor suppression activity of the polypeptides of the present
invention has been monitored by their ability to reduce or inhibit
the growth of prostatic adenocarcinoma both in-vivo and in-vitro.
Those results are summarized below.
Studies were carried out using PC-3 human prostate
adenocarcinoma line, which can be maintained both in vivo as a
xenograft in nude mice and in vitro as a cell line. In addition, a
rat Dunning Mat LyLu prostate tumor, which is a pre-eminent animal
model for the study of CaP, was also used. The Dunning tumor is a
fast growing, poorly differentiated, transplantable tumor, which can
be maintained both in-vivo in the Copenhagen rat and in-vitro as a
cell line.
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The following examples are offered by way of illustration and not by
way of limitation.
EXAMPLE 1
PREPARATION OF rHuPSP94 (SEQ ID NO: 2) and polypeptides (SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6)
Recombinant HuPSP94 was cloned and expressed in Pichia pastoris, and
then purified and characterized as follows.
Materials
DEAE-cellulose (DE52) was purchased from WhatmanT' (Fairfield, New-
Jersey). Dialysis membranes and the electro chemiluminescence (ECLTM)
detection kit were purchased from Biolynx Canada(Pierce Inc.). Broad-range
molecular weight markers and Econo-pack columns fitted with flow adapters
were purchased from Bio-Rad Labs Ltd (California). PelliconT' device was
purchased from Millipore (Massachusetts). Tris-HC1 was obtained from ICN.
MES ((2-[N-Morpholino]ethanesulfonic acid) hydrate)was obtained from Sigma.
Swine anti-rabbit IgG alkaline-phosphatase conjugates was purchased from
DAKO (Denmark). Pichia Pastoris expression Kit version G was from
Invitrogen(Carlsbad, California). Non-Radioactive High Prime DIG labeling
kit was purchased from Boehringer Mannheim (Indianapolis, Indiana). The
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-
sulfophenyl)-2H-tetrazolium, inner salt) assays were performed using Cell
Titer Aqueous Non radioactive cell proliferation assay kit from Promega
(Madison, Wisconsin). MRX microtiter plate reader was from Dynex
technologies (Chantillly, Virginia). Rabbit polyclonal antiserum against
PSP94 was a gift from the late Dr. A. Sheth. All primers were synthesized
by Procyon Biopharma Inc. London, Ontario, Canada.
Cell line and cell culture
P. pastoris host strain GS115 (his4) and all Pichia related products
were obtained from Invitrogen. PC-3 (ATCC-# CRL 1435) cell line was
obtained from the American Type Cell Culture (ATCC) and maintained in OPTI
MEM (minimum essential media) with 10 % fetal bovine serum (FBS). All cell
culture products were obtained from GIBCO BRL.
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Cloning
TA cloning vector (pCR TM 2.1) containing human PSP94 cDNA including
a 20 amino acid leader sequence described previously (Baijal-Gupta, M., et.
al., J. Endocrinol., 165:425-433, 2000) was used to amplify human PSP94
without its leader sequence using appropriate primers. The primers for the
polymerase chain reaction (PCR) were designed to contain an EcoRI
restriction sites at either end. The 5' primer used was 5'-GGG AAG AAT TCT
CAT GCT ATT TCA TA-3' (SEQ ID NO: 7) and the 3' primer, 5'-TGG ATA TCT GCA
GAA TTC GGC-3' (SEQ ID NO: 8). The +1 start site for PSP94 (at a Serine
residue) has been underlined in the 5' primer described above.
The PCR included 1 cycle of 12 minutes at 94 C, followed by 25
cycles of 1 minute at 94 C, 1 minute at 55 'C, 1 minute at 72 'C and a
final step of 1 cycle of 10 minutes at 72 C. PCR amplification of the
product was performed using BM Expand" High Fidelity PCR System. The
product was run on a 1.5 % agarose gel and the appropriate PCR product was
isolated using Pharmacia SephaglasTM Kit (Bandprep) . Subcloning of the
PSP94 insert was performed in pPIC9 vector (Invitrogen). The EcoRI enzyme
was used for the restriction digestion of both the plasmid and the PCR
products (thus removing PSP94 signal sequence) followed by ligation and
transformation, using DH5a cells. The isolated clones were selected for by
ampicillin resistance and inserts were identified by restriction mappings.
The constructs were sequenced (Robart's sequencing service, London,
Ontario) to identify PSP94 insert with a correct sequence as well as proper
orientation and reading frame.
Screening for Clones Expressing rHuPSP94
For Pichia pastoris transformation, the spheroplast method was used
according to manufacturer's instructions (Invitrogen) using GS115 and KM71
yeast strains. Plasmid pPIC9 with or without the PSP94 insert were
linearized using Sall restriction enzyme. Transformed colonies were
screened and selected for their ability to produce their own histidine,
hence survived on media without histidine. All GS115 transformants scored
as Mut`, whereas all KM71 colonies, which did not grow well in the liquid
culture, scored as Muts. Hence a number of GS115 clones were screened for
production of the highest levels of rHuPSP94 expression.
About a hundred clones were selected and grown into 2 ml of culture
media until an optical density at 600 nm (OD600) of approximately 6 was
reached. Total DNA was isolated for rapid dot
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blot analysis in order to detect multiple integrations by Southern blot
that would possibly correspond to high rHuPSP94 expressing clones. Two
hundred microliters of each culture specimens were denatured and blotted
(in duplicate) to a positively charged nylon membrane, placed in a dot blot
apparatus. The membrane was subsequently air-dried. The membrane was
soaked between two sheets of WhatmanTM 3MM paper for 15 minutes in a
solution containing 50mM ethylenediaminetetraacetic acid(EDTA), 2.5 % beta-
mercaptoethanol (BME), pH 9, followed by an incubation of 24 hours at 37 C
with 1 mg/ml Zymolyase 100T, 5 minutes in 0.1 N NaOH, 1.5 M NaCl, 0.015 M
sodium citrate pH 7 and two 5 minutes incubation in 2x saline-sodium
citrate (SSC). Finally the membrane was baked at 80 C for 45 minutes and
exposed to ultraviolet light (W) for 15 minutes. Human PSP94 cDNA probe
was labeled with the non-radioactive High Prime DIG labeling kit
(Boehringer Mannheim) and was used for hybridization. Hybridization with
digoxigenin labeled cDNA probe (25ng/ l) was done for 2 days at 42 C in
Sodium dodecyl sulfate(SDS) buffer (SDS 7 % (w/v); formamide 50 % (v/v); 5
X SSC; 50 mM sodium phosphate, pH 7.0; N-lauroyl-sarcosine 0.1 t (w/v)) and
blocking reagent, CSPD 2 % (w/v) (Boehringer Mannheim) was used as the
chemiluminescence substrate. All digoxigenin (DIG) labeling procedures
were performed according to the manufacturer's instruction. Detection was
performed using the HyperfilmTM-ECLT' product (Amersham Life Science Inc.
Arlington Hts, Illinois).
The clone with the highest signal intensity was used for all flasks shaken
cultures.
Optimization of the Expression of the Protein in Flask Shaken Cultures
A clone containing the PSP94 construct was selected for high
expression of the protein. Colony was grown in 25ml of basal minimum growth
media (BMG) until an OD600 between 2 and 6 was obtained. This clone was
further amplified in Baffled Erlenmeyer flasks in a volume of 1 liter of
BMG media until the OD600 reached approximately between 2.0 to 6Ø The
culture was centrifuged for 15 minutes at 2500 X g and the pellet was
collected. The induction phase (i.e., induction of expression of rHuPSP94)
was carried out by inoculating the cell pellet in basal minimum media
(BMM). Growth was performed in Baffled flasks for 6 days, as recommended
by Invitrogen. The volume of BMM added varied according to the size of the
pellet collected. Five milliliters of 100 % methanol were added for each
liter of culture. This was performed each day, around the same time, to a
final
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concentration of 0.1 s of methanol. A plasmid without the PSP94 insert
served as a negative control.
To determine the optimum time for harvesting rHuPSP94 secreted in
the cell culture media, aliquots were taken every 24 hours for 6 days,
starting from the first day of induction. Levels of rHuPSP94 protein
expression were determined by measuring OD600 and by performing a 15 % SDS-
PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) stained
with Coomassie Brilliant blue or by Western blot analysis using polyclonal
antibody against PSP94.
Sample Preparation
Culture supernatant of clone showing the highest rHuPSP94
expression, post-induction (e.g., after 96 hours), was centrifuged at 2500
X g for 20 minutes. The supernatant was filtered through a 0.8 pm filter
and concentrated approximately 10-fold using a Pellicon unit (Millipore).
The filtered supernatant was dialyzed against 0.05 mM Tris-HC1 buffer, pH
8.0, using a 3500 molecular weight cut-off membrane. An aliquot of the
dialyzed supernatant was analyzed by SDS-PAGE and Western blot analysis and
the rest was submitted to further purification.
Culture Conditions for Fermentation
Fermentation was carried out at the Institute for Biological
Sciences, National Research Council (NRC) (Ottawa, Ontario Canada),
following manufacturer's instruction (Invitrogen). For example, a
fermentation procedure was initiated by inoculating 7.5 liter of media with
625 ml of a starting culture. The growth phase was carried out for
approximately 2 days in BMG media until the OD600 reached approximately
0.5. The induction phase was initiated by the addition of methanol (100
%), according to the manufacturer's instructions (Invitrogen). The culture
was harvested after 95 hours (i.e., after induction with methanol for 67
hours). The final volume of the culture was approximately 13.5 liters.
Sample Preparation from Fermentation Culture
The large cell mass was removed by centrifugation. The cell free
media collected (9 liters) was further clarified using a 0.2 m filtration
unit (PelliconTM). The remaining 8.5 liters containing secreted rHuPSP94 was
tested for protein expression and stored at -20 C for further isolation
and purification of the protein.
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Protein Estimation
The amount of rHuPSP94 protein secreted in the culture supernatant
from the flask shaken and the fermentation process was obtained based on
estimates of band intensities of samples compared to band intensities of a
standard curve obtained by loading known quantities of pure lyophilized
PSP94 on a SDS-PAGE. The initial estimate for rHuPSP94 at each step of
purification was determined by OD at 280 nm. Quantification of total
protein content at the final steps of purification was done by the BCA
(bicinchoninic acid) method, using bovine serum albumin (BSA) as standard.
Lyophilization
Samples of purified rHuPSP94 were dialyzed against deionized water
using a 3000 molecular weight cut-off membrane and were lyophilized.
SDS-PAGE
SDS-PAGE was performed using acrylamide at a final concentration of
15 % for the separating portion of the gel and acrylamide at a final
concentration of 5 % for the stacking portion of the gel. The gel
contained 0.1 % SDS and was performed under reducing conditions. Broad-
range molecular weight markers were used for the estimation of molecular
weight of the protein. Proteins were stained with Coomassie Brilliant Blue
R-250.
Western Blotting
For immunoblotting, Mini Trans-Blot Electrophoretic Transfer Cell
(Bio Rad) was used with HybondTM-C super membrane (Amersham) and 85mm
blotting papers. Protein samples (0.4 jig) were loaded and separated on SDS-
PAGE, as described earlier. Proteins were transferred to the membrane for 2
hours at 4 C, using transfer buffer (25 mM Tris, 192 mM Glycine, pH 8.3
and 20 % methanol) and a transfer unit set at 200 milliamperes (mAmp).
Membranes were blocked overnight by incubation in 2 % (w/v) non-fat dry
milk (skim milk) disolved in tris buffer saline (TBS: 500 mM NaCl, 20 mM
Tris-HC1, pH 7.5) at room temp (RT). Membranes were washed three times
with TBS containing 0.02 % (v/v) Tweenm-20 (this buffer is named TTBS).
Membranes were subsequently incubated for 2 hours at RT with anti-PSP94
antibody (1:2000 dilution) diluted in TTBS containing 2 % skim milk.
Membranes were washed twice with TTBS (5 minutes each washing), and
incubated at RT with a secondary antibody (i.e., swine anti-rabbit antibody
HRP conjugated) (1:5000 dilution) diluted in TTBS. Membranes were washed
twice with 0.02 % TTBS (5 minutes each washing). Blots were developed
using the ECLTM detection system,
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according to manufacturer's instructions, using the Super Signal Substrate,
and exposed to a HyperfilmTM ECLTM from Amersham LS for 5 to 20 seconds.
Pre-stained molecular weight markers were used for molecular weight
estimation.
Purification of rHuPSP94 using DE52 Column Chromatography
Following removal of P. pastoris cells from the fermentation
culture, supernatant was concentrated approximately ten fold, dialyzed and
subjected to anion exchange chromatography. A DE52 column having a bed
volume of approximately 40 ml (2.5 cm internal diameter (id) X 8 cm
height(h)) was equilibrated with 0.05 M Tris-HC1, pH 8.0 (equilibrating
buffer). The sample (25 ml) containing 15 to 20 mg of rHuPSP94 protein was
applied to the DE52 column at a flow rate of 1 ml/minute.
Impurities were removed from the column by washing it with 40 to 50
ml of the equilibrating buffer, and monitoring the absorbance at 280 nm.
This step was followed by the addition of 100 to 150 ml of 0.05 M Tris-HC1,
pH 6.5 to the column until the pH of the wash reached approximately 6.5.
The column was further washed with 100 to 150 ml of 0.05 M MES-acetate
buffer, pH 6.5, until the absorbance at 280nm approached zero. Finally
rHuPSP94 was eluted from the column with 0.05 M MES-acetate buffer, pH 5Ø
Peak fractions were characterized by absorbance at 280 nm, followed by SDS-
PAGE and Western blot analysis as described above. Fractions with high
absorbance at 280 nm values (0.5 to 1.8) were pooled and dialyzed against
water or PBS for storage at -20 C and/or lyophilization.
Amino acid Composition
Amino acid analysis of the DE52 purified flask shaken culture and
fermentation cultures was carried out. The Perkin Elmer Biosystems
Derivatizer-Analysis system was used with Spheri-5 PTC C-18 5 column and
UV detection at OD254.
Mass Spectral analysis
PSP94 derived polypeptides were synthesized, were found to be in
accordance with the required specifications and were analyzed by mass
Spectral Analysis. Mass spectrometry analysis of polypeptide 7-21 (SEQ ID
NO: 4), PCK3145 (SEQ ID NO: 5) and polypeptide 76-94 (SEQ ID NO: 6) are
represented in figures 1, 2 and 3 respectively.
Polypeptide samples were analyzed using the PerSeptive Biosystems
(Framingham, MA), with Voyager-DE MALDI-TOF mass
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spectrometer using 337 nm light from a nitrogen laser. About 12 to
50 scans were averaged for each analysis.
Purified samples from the flask shaken culture and
fermentation culture were analyzed using the PerSeptive Biosystems
(Framingham, MA), with Voyager-DE MALDI-TOF mass spectrometer using
337 nm light from a nitrogen laser. About 50 scans were averaged for
each analysis. A sample from the native PSP94 was also analyzed
under similar conditions for comparison.
EXAMPLE 2
IN-VITRO EFFECT OF rHuPSP94 ON PC-3 CELLS
(MTS ASSAY)
The biological activity of the rHuPSP94 was determined by its
growth inhibitory effect on human prostate cancer cells PC-3. Cell
proliferation was monitored on PC-3 cells using the MTS/PMS
(phenazine methosulfate) kit (Promega), which primarily measures
mitochondrial activity of live cells. The basic principle of this
method involves the fact that the mitochondrial enzymes of the live
cells metabolize the MTS/PMS dyes forming a brown colored precipitate
which can be measured as optical density (OD) by absorption at 490 nm
in a spectrophotometer. Therefore, the OD values are proportional to
the number of living cells. In addition, monitoring of cell
morphology was also performed. Cell morphology would be indicative
of their health status. For example, viable cells would appear
adherent and spread out whereas dead cells would be in suspension in
the media and would appear granular and round.
Results of in vitro effect of rHuPSP94 on PC-3 cells measured
by MTS assay are summarized in table 2, below. PC-3 cells (ATCC, Lot
AT06) used in these experiments were at a passage number lower or
equal to 70 (n >_ 70). Cells were seeded in Costar 96 well cell
culture flat bottom plates in RPMI supplemented media containing 50
g/ml of bovine serum albumin (BSA) and 0.1 M FeSO4. Peptide was
diluted in the same media. Cells were continuously exposed to the
polypeptides of the present invention for 72 hours without changing
media. Native PSP94 or rHuPSP94 concentrated two fold were directly
added to wells and diluted to 1X in order to minimize cell
manipulation and avoid detachment.
The evaluation of growth inhibitory effect of rHuPSP94 on PC-3
cells indicated a substantial reduction in cell numbers (i.e.,
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viability) ranging from 37 % to 57 % reduction at concentrations of
80 and 120 g/ml of rHuPSP94 respectively. This effect was observed
in 3 out of 4 experiments (Table 2). Results of trypan blue
exclusion test demonstrated a cell viability of 62 % at 80 g/ml.
TABLE 2
Experiment Sample % Viability (control = 100%) ( g/ml)
no. 40 60 80 120
1 rHuPSP94 72 78 58 43
2 rHuPSP94 63 63 63 68
3 rHuPSP94 95 85 78 ND
4 rHuPSP94 100 52 62 60
5 rHuPSP94 100 98 90 52
Sample % Viability (control = 100%) ( g/ml)
5 10 20 40 80
rHuPSP94 98 84 78 70 55
rHuPSP94 92 95 80 71 59
rHuPSP94 89 69 79 68 65
EXAMPLE 3
IN-VITRO EFFECT OF rHuPSP94 ON PC-3 CELLS
([3H]-THYMIDINE UPTAKE ASSAY)
The in vitro growth inhibition effect of rHuPSP94 was assessed
using [3H]-Thymidine uptake assay. [3H]-Thymidine uptake assay
involves [3H]-Thymidine incorporation into cellular DNA of actively
proliferating cells. It measures the proliferative index of the cells
versus the MTS assay, which quantifies the number of lived cells
following treatment. Cells were seeded in Costar 96 well cell culture
flat bottom plates in RPMI supplemented media containing 50 g/ml of
bovine serum albumin (BSA) and 0.1 pM FeS09. PC-3 cells were exposed
to various concentrations of rHuPSP94 for 72 hours and during the
final 16 hours of incubation cells were pulsed with 1 pCi of [3H]-
Thymidine. The radioactivity in each well of the plate is counted by
a beta-counter and is expressed as total counts per minutes (cpm).
Results of in vitro effect of rHuPSP94 on PC-3 cells using the 3[H]-
Thymidine uptake assay are summarized in Table 3 and are expressed as
percentage of radioactivity measured for treated-cells relative to
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the radioactivity measured for non-treated cells (for which [3H]-
thymidine uptake value was set at 100 %).
Results indicated a 65 % reduction in the percentage of cells
incorporating [3H]-thymidine following treatment with rHuPSP94 at a
concentration of 80 g/ml for 72hrs, compared to the non-treated
control. Results of a 65 % reduction in [3H]-thymidine uptake may
also be an indication of a 65 % reduction in cell proliferation.
Comparison was performed between [3H]-Thymidine uptake assay
and the MTS assay, in order to evaluate their relative sensitivity.
An additional plate was set aside for MTS assay and treated in
parallel with the same lot (i.e., batch) of rHuPSP94 as the one used
for the [3H]-thymidine uptake assay. Result obtained for the MTS
assay demonstrated a 35 % reduction in cell viability (65 % cells
remaining viable) following treatment with rHuPSP94 at a
concentration of 80 g/ml, indicating that the [3H]-Thymidine uptake
assay, which was able to measure a 65 % reduction in cell
proliferation, may be more sensitive than the MTS assay.
TABLE 3
Experiment Sample 3[H]-Thymidine Uptake (% of control)
no. ( g/ml)
5 1.0 20 40 80
1 rHuPSP94 94 101 98 79 35
1 native PSP94 97 98 100 98 77
EXAMPLE 4
IN-VITRO EFFECT OF DECAPEPTIDE AND OTHER POLYPEPTIDE ON
PC-3 CELLS
The synthetic decapeptide (SEQ ID NO: 3) has been shown herein
to mimic the biological activity of native PSP94 (nPSP94)(SEQ ID NO:
1) and therefore its effect on the PC-3 cells was studied in
clonogenicity assay (colony formation). Cells were seeded in Costar
96 well cell culture flat bottom plates in RPMI supplemented media
containing 50 g/ml of bovine serum albumin (BSA) and 0.1 pM FeSO4.
Clonogenicity was evaluated for PC-3 cells grown in the presence of
various concentration of the decapeptide after 9 days of culture
(Figure 4a). A parallel experiment was performed with various
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concentration of nPSP94 using the same experimental conditions
(Figure 4b). Other experiments evaluating clonogenicity was
performed with the decapeptide (Figure 5a) or nPSP94 (Figure 5b)
after 21 days of culture as well as after 10 days of culture (Figure
6a: Decapeptide and Figure 6b: nPSP94).
Referring to Figures 4 to 6, the decapeptide (SEQ ID NO: 3)
had a similar inhibitory action as nPSP94 (SEQ ID NO: 1) on in-vitro
PC-3 cells studied. Results indicated a 40 % decrease in colony
number for cells incubated with the decapeptide (SEQ ID NO: 3) at a
concentration of 1 g/ml. A decrease in colony number of up to 60 %
was observed for the decapeptide (SEQ ID NO: 3) at a concentration of
10 g/ml.
EXAMPLE 5
DNA FRAGMENTATION ASSAY
Cell apoptosis result in DNA fragmentation can be evaluated by
the presence of a DNA ladder visualized when DNA is run on a 1.2 %
agarose gel. DNA ladder assay (apoptosis assay) was performed
following exposure of PC-3 to various concentrations of the
polypeptides for 72 hours. The polypeptides that were used in this
particular experiment are polypeptide 7-21 (SEQ ID NO: 4),
polypeptide PCK3145 (SEQ ID NO: 5) and polypeptide 76-94 (SEQ ID NO:
6). Visualization of DNA isolated and run on 1.2 % agarose gel,
demonstrated that every polypeptides tested induced a DNA laddering
effect characteristic of apoptosis. This effect was especially
evident following treatment with PCK3145 (SEQ ID NO: 5), which is
illustrated by figure 7. Lane 1 of the gel illustrated in figure 7
represents a lambda Hindlll digest standard. Lane 2 of the gel
illustrated in figure 7 represents DNA laddering effect obtained for
doxorubicin-treated cells. Lane 3 of the gel illustrated in figure 7
represents DNA laddering effect obtained for cells incubated with 40
g of nPSP94. Lane 4 of the gel illustrated in figure 7 represents
DNA laddering effect obtained for cells incubated with 20 g of
nPSP94. Lane 5 of the gel illustrated in figure 7 represents DNA
laddering effect obtained for cells incubated with 22.5 M of PCK3145
(SEQ ID NO: 5). Lane 6 of the gel illustrated in figure 7 represents
DNA laddering effect obtained for cells incubated with 45 M of
PCK3145 (SEQ ID NO: 5).
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EXAMPLE 6
APOPTOSIS ASSAY BY ELISA PLUS
The three polypeptides (SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO 6)
and native PSP94 used here as a positive control were tested in ELISA plus
assay to measure cell death through apoptosis. Briefly, the ELISA plus
assay is a sandwich enzyme immunoassay able to measure mono- and
oligonucleosomes present in the cytoplasmic fraction of cell lysate using
two antibodies, one directed against DNA and the other directed against
histones. The apoptotic cell death is characterized by activation of
endogenous endonucleases (e.g., calcium- and magnesium-dependant), which
cleave double-stranded DNA at the most accessible internucleosomal linker
region, generating mono- and oligonucleosomes. The enrichment of mono- and
oligonucleosomes in the cytoplasm of the apoptotic cells is due to the fact
that DNA degradation occurs several hours before plasma membrane breakdown.
Four thousand cells were seeded in Costar 96 well cell culture flat
bottom plates in RPMI supplemented media containing 50 g/ml of bovine
serum albumin (BSA) and 0.1 M FeSO4. The PC-3 cells were treated with
various concentrations (22.5 M to 90 M) of polypeptides for 72 hours.
Apoptosis assay was done as per manufacturer's instructions using the
ApopTagTM kit (Boeringher Mannheim).
Results presented in figure 8, indicate a dose dependent increase in
the apoptotic cell death effect was observed for every polypeptides used
(SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO 6). Polypeptide PCK3145 (SEQ ID
NO: 5) was more potent than the other polypeptides at 90 M concentration
(Figure 8).
EXAMPLE 7
INHIBITION OF CELL-GROWTH BY PSP94 POLYPEPTIDES
(Figures 9 to 11)
Biological activity of the polypeptides as set forth in SEQ ID NO: 4, SEQ
ID NO: 5 and SEQ ID NO 6 was determined by their growth inhibitory effect
on human prostate cancer cells PC-3. Native PSP94, rHuPSP94, polypeptide
22-36 and PB111 polypeptide (scrambled polypeptide) were also included in
this experiment as controls. Cell proliferation assay was performed on
either PC-3 cells or normal fibroblasts (used here as control) using the
MTS/PMS kit (Promega).
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Four thousand cells (Figure 9 and 10) or three thousand (figure 11)
cells were seeded in Costar 96 well cell culture flat bottom plates
in RPMI supplemented media containing 50 g/ml of bovine serum
albumin (BSA) and 0.1 M FeS04. In addition, monitoring of cell
morphology was also performed.
Results of these experiments are shown in figures 9 to 11. No
cell inhibitory effect was observed following incubation of
fibroblasts with various polypeptide concentrations (from 10 to 90
M) for 72 hours (Figure 9). However, a significant growth
inhibition was observed for polypeptides as set forth in SEQ ID NO: 4
and SEQ ID NO: 6 and more importantly with polypeptide PCK3145 (SEQ
ID NO: 5) (Figure 10). Another experiment was performed using PCK3145
and polypeptide 22-36 at various concentrations on PC-3 cells, grown
in OPTI-MEM media. In figures 9 to 11, the percentage of growth
inhibition given for treated cells is evaluated relative to non-
treated control cells for which a value of 100 % cell survival is
given.
EXAMPLES 8 & 9
IN-VIVO EXPERIMENTS (Figures 12 & 13)
Studies MLL-1 and MLL-2 were performed as follows; on day 0,
male Copenhagen rats were injected subcutaneously with 5 x 105 Mat
LyLu cells per rat. These cells were derived from cultures of Mat
LyLu cell line grown in RPMI media containing 10 % (v/v) of fetal
calf serum in logarithmic phase of growth. Cells were harvested from
the culture flasks by trypsinization, were centrifuged at 1200
rotation per minute (rpm) and washed three timed with Hanks balanced
salt solution (HBSS). Following washing, cells were counted and
adjusted to a concentration of 5 x 10'3 cells/ml in HBSS. A 0.1 ml
volume of tumor cell inoculum containing 5 x 105 cells was
administered subcutaneously into the flank region of each rat. Three
days after tumor cell implantation (i.e., inoculation), animals were
treated daily by a subcutaneous injection of the desired polypeptide
until day 13.
Experiments illustrated in figure 12 show the anti-tumor
efficacy validation of rHuPSP94 against Mat LyLu (MLL) tumor
implanted in nude mice (Protocol based on S. Garde et al.; The
Prostate, 22: 225-233, 1993).
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For study MLL-1 (Figure 12), tumor-implanted nude mice were
separated in different groups, each receiving various amount of
rHuPSP94 or control reagents. The different groups used in these
experiments are illustrated below. Each group contained 8 mice.
Group 1: Negative control: PBS subcutaneously (s.c.)
Group 2: Positive control: Doxorubicin at 5mg/kg intraveanously
(i.v.) single bolus on day 3
Group 3: rHuPSP94 at 1 g/kg/day (s.c.)
Group 4: rHuPSP94 at 10 g/kg/day (s.c.)
Group 5: rHuPSP94 at 100 g/kg/day (s.c.)
A schematic of inoculation is illustrated below;
(Tumor cell implantation (T.C.I.), treatment (Tx), measurement (M),
day (D)).
M== M1 M M
DO 3 I57 I~9 ill D13 D14
t t t
T.C.I. Tx daily Tx Terminate
Experiments illustrated in study MLL-2 show the anti-tumor
efficacy validation of rHuPSP94 against Mat Ly Lu (MLL) tumor
implanted in severe combined immunodeficiency (SCID) mice (Protocol
based on S. Garde et al.; The Prostate, 22: 225-233, 1993).
For study MLL-2 (figure 13), tumor-implanted Scid mice were
separated in different groups each receiving various amounts of
rHuPSP94 or control reagents. The different groups used in these
experiments are illustrated below. Each group contained 8 mice.
Group 1: Negative control: PBS (s.c.)
Group 2: Positive control: Doxorubicin at 5mg/kg i.v. single bolus on
day 3
Group 3: rHuPSP94 at 1 g/kg/day (s.c.)
Group 4: rHuPSP94 at 10 g/kg/day (s.c.)
Group 5: rHuPSP94 at 100 g/kg/day (s.c.)
A schematic of inoculation is illustrated below;
(Tumor cell implantation (T.C.I.), treatment (Tx), measurement (M),
day (D)).
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MN MN M M
D~14
DO 3 1~7 D9 D11 D13
I * I
T.C.I. Tx daily 10 Tx Terminate
Results of those two studies indicate a difference in tumor
size and growth in Nude vs SCID mice. The tumors grew slower and were
smaller in SCID mice. This may be due to some specific factors
controlling tumor growth in this mouse strain. Results also show a
significant tumor reduction in mice injected with Doxorubicin
(positive control). For example, tumor weight reduction in Nude mice
(study MLL-1) injected with Doxorubicin was 48 % (p=0.006)(p values
measured by unpaired Student's t-test at p<0.05 as cut-off limit).
Tumor weight reduction in SCID mice (study MLL-2) inoculated with
Doxorubicin was 82 % (p=0.002) (p values measured by unpaired
Student's t-test at p<0.05 as cut-off limit). Results indicate also
a significant tumor reduction in mice treated with rHuPSP94 at a
concentration of 1 g/kg/day. For example, tumor weight reduction in
Nude mice (study MLL-1) treated with rHuPSP94 at a concentration of 1
g/kg/day was 26 % (p=0.042) (p values measured by unpaired Student's
t-test at p<0.05 as cut-off limit). Tumor weight reduction in SCID
mice (study MLL-2) treated with rHuPSP94 at a concentration of 1
g/kg/day was 65 % (p=0.010) (p values measured by unpaired Student's
t-test at p<0.05 as cut-off limit).
EXAMPLE 10
IN-VIVO EXPERIMENT USING PC-3 CELL LINE
(Figure 14)
PC-3 human prostate tumor was obtained from ATCC (ATCC
1435). PC-3 cells were grown in RPMI media containing 10 % (v/v)
of fetal calf serum and were harvested in the logarithmic phase of
growth by trypsinization. Cells were centrifuged at 1200 rotation
per minute (rpm) and washed three timed with Hanks balanced salt
solution (HBSS). Following washing, cells were counted and
adjusted to a concentration of 1 x 10' cells/ml in HBSS. A 0.1 ml
volume of tumor cell inoculum containing 1 x 106 cells was
administered subcutaneously into the two opposite flank region of
each Nude mouse (Nu/Nu, BALB/c background). Tumor growth was
monitored for approximately 18 days. Once tumor growth has been
established (volume of tumor reached a volume of 50 mm 3) treatment
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with rHuPSP94 (SEQ ID NO: 2) was initiated and was performed once a
day for 14 days by the subcutaneous route. Based on the assigned
treatment groups illustrated in table 4.
TABLE 4
Treatment Test control Dose Level Dose No. of
group articles ( g/kg/day) concentration animal
( g/mg)
1 Negative PBS 0 0 8
control
2 Positive Doxorubicin 5000 2500 8
control
3 rHuPSP94 1 0.5 8
4 rHuPSP94 10 5 8
5 rHuPSP94 100 50 8
6 rHuPSP94 1000 500 8
Results of this experiment (Figure 14) demonstrated tumor
growth reduction in the group of mice treated with rHuPSP94 at a
dosage level of 1 g/kg body weight per day. This reduction was
similar to that observed for Doxorubicin (given at 5 mg/kg/day) which
is a chemotherapeutic agent used as reference gold standard.
EXAMPLE 11
IN-VIVO EXPERIMENT USING PC-3 CELL LINE
(Figures 15-17)
PC-3 human prostate tumor (ATCC 1435) obtained from ATCC was
implanted bilateraly into nude mice and tumor growth was monitored
for approximately 18 days. PC-3 cells were injected once
subcutaneously into each flank of the mice. Once tumor growth has
been established (i.e., volume of tumor reached 0.25 to 0.50 cm3)
the treatment with decapeptide (SEQ ID NO: 3), native PSP94 (SEQ ID
NO: 1) and control scrambled polypeptide PB111 was initiated and
was performed once a day for 14 days by the subcutaneous route
based on the treatment groups (randomly assigned) illustrated in
table 5.
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TABLE 5
Treatment Test and control Dose Level Dose No. of
groups articles ( g/kg/day) concentration animal
( g/m9)
l(Negative PBS 0 0 4
control)
3 Decapeptide 1 0.5 4
(SEQ ID NO: 3)
4 Decapeptide 10 5 4
(SEQ ID NO: 3)
Decapeptide 100 50 4
(SEQ ID NO: 3)
6 Decapeptide 1000 500 4
(SEQ ID NO: 3)
7 Native PSP94 1 0.5 4
(SEQ ID NO: 1)
8 Native PSP94 10 5 4
(SEQ ID NO: 1)
Native PSP94 100 50 4
(SEQ ID NO: 1)
11 Native PSP94 1000 500 4
(SEQ ID NO: 1)
12 Scrambled 1 0.5 4
polypeptide(PB111)
13 Scrambled 10 5 4
polypeptide(PB111)
14 Scrambled 100 50 4
polypeptide(PB111)
Scrambled 1000 500 4
polypeptide(PB111)
Figure 15 represents results obtained for tumor-implanted nude
5 mice treated with the decapeptide (SEQ ID NO: 3) compared to a non-
treated control. Figure 16 represents results obtained for tumor-
implanted nude mice treated with scrambled polypeptide PB111 compared
to a non-treated control. Figure 17 represents results obtained for
tumor-implanted nude mice treated with native PSP94 (SEQ ID NO: 1)
10 compared to a non-treated control. Results of these experiments
(Figures 15-17) indicate a significant (p<0.05) tumor growth
reduction in mice treated with the decapeptide (SEQ ID NO: 3) at a
dosage level of 10 pg/kg body weight per day.
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EXAMPLE 12
MANUFACTURING AND PREPARATION OF POLYPEPTIDES
PSP94 derived polypeptides including PCK3145 (SEQ ID NO: 5)
were synthesized using the FMOC and BOC solid phase polypeptide
synthesis method (Merrifield, B., Science, 232: 341-347, 1986).
Polypeptides were analyzed in order to determine their identity by
Mass Spectral Analysis. Polypeptide samples were analyzed using the
PerSeptive Biosystems (Framingham, MA), with Voyager-DE MALDI-TOF
mass spectrometer using 337 nm light from a nitrogen laser. About 50
scans were averaged for each analysis. A sample from the native PSP94
was also analyzed under similar conditions for comparison.
Polypeptides were weighed on a Mettler AE 163 micro-balance. The
measurements were to nearest 0.1 mg. The polypeptides were
reconstituted in 10 mM PBS pH 7.3 to a final concentration of 1 and 5
mg/ml. The polypeptides dissolved relatively well and were filter
sterilized through a 0.2 M syringe filter. Aliquots of 2 ml/tube
were made and stored at -80 C.
The pH of the polypeptides was measured after reconstitution to
ensure that possible differences in pH would not be a factor of
variation. The pH values of each solution were taken at three
concentrations: neat, 100 g/ml and 12.5 g/ml. The pH range was
approximately from 7.0 to 7.5. This did not make a significant
difference in the outcome of the test as cells survive very well
within this pH range. To change the concentrations to molar values,
the approximate volume of the 1 mg/ml stocks were diluted in PBS pH
7.3. All stocks were made to contain 450 M polypeptide solutions.
When fresh stocks of polypeptide were to be reconstituted, it was
done directly to 450 M concentration in PBS pH 7.3.
After our initial screening and confirmation of the inhibitory
activity of the polypeptide on the growth of the PC-3 cells, a GMP
manufactured polypeptide was tested. This polypeptide was weighed and
dissolved in PBS and 2 mg/ml stock solution was prepared, sterile
filtered through a 0,2 pm syringe filter and stored at in -80 C.
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EXAMPLE 13
EFFECT OF PCK3145 ON IN-VITRO PC-3 CELLS
(MTS ASSAY (Figures 18-21))
PCK3145, manufactured as set forth in example 12, was evaluated
as a lead candidate product in tumor growth inhibition.
The biological activity of PCK3145 was determined by its growth
inhibitory effect on the human prostate cancer cell line PC-3 using
the MTS/PMS kit (Promega). This assay measures the mitochondrial
activity of the live cells. The basic principle of this method
involves the fact that the mitochondrial enzymes of the live cells
metabolize the MTS/PMS dyes forming a brown colored precipitate which
can be measured as optical density (01D) by absorption at 490 nm in a
spectrophotometer. Therefore, the OD values are proportional to the
number of living cells.
In addition, a visual observation of the cells was also done to
check the cell morphology, which could also be indicative of cell
growth. The following conditions for MTS assay were used: PC-3
(ATCC, Lot AT06), passage number n >_ 70, cell line adapted to grow in
serum-free OPTI-MEM and in RPMI supplemented with BSA (50 pg/ml) and
Ferrous Sulfate (0.1 M), continuous exposure for up to 72 hours
without changing media (i.e., adding PCK3145 at 2X concentration
directly to wells and diluting it 1:2 to 1X to minimize cell
manipulation and avoid detachment). As indicated in Figure 18,
PCK3145 was assessed at the following concentrations: 12.5, 25, 50,
100, 200, 300 and 400 pg/ml on PC-3 cells (ATCC) grown in
supplemented media. The MTS tests were repeated 5 times and a dose
dependent inhibitory effect on the growth of PC-3 cells was
consistently reproducible demonstrating approximately 40 % cell
growth inhibition at the highest PCK3145 concentration of 400 .tg/ml.
With the availability of GMP (good manufacturing practice)
grade polypeptide the MTS assays were repeated to check the
reproducibility and cytotoxicity against PC-3 cells. In parallel PC-
3 cells were also treated with the native PSP94 as a reference
positive control and with no treatment (negative control, i.e.,
cont.). Figure 19 shows the results of the MTS assay where 4000
cells were seeded and exposed to PCK3145 (GMP grade) for 48 hours. A
30 % growth inhibitory effect was observed following treatment with
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PCK3145 at 500 g/ml. This effect was increased to approximately 40 %
after 72 hours of exposure (Figure 20). In a repeat experiment a 48
hours exposure to the polypeptide at 500 gg/ml resulted in only 20-
22 % growth inhibition, however this effect increased to 30 % after
72 hours exposure (Figure 21). Despite assay to assay variability
reflected by the state of cell growth in vitro, polypeptide PCK3145
exhibited a significant cell growth inhibition.
EXAMPLE 14
EFFECT OF PCK3145 ON IN-VITRO PC-3 CELLS
[3H]-THYMIDINE UPTAKE ASSAY (Figures 22-24)
[3H]-Thymidine uptake assay involves [3H]-Thymidine
incorporation into cellular DNA of actively proliferating cells.
[3H]-Thymidine uptake assay measures the proliferative index of the
cells versus the MTS assay, which quantifies the number of lived
cells following treatment. The anti-proliferative effects of PCK3145
and two other synthetic polypeptides derived from the amino and
carboxy terminus ends of PSP94 (SEQ ID NO: 4 and NO: 6, respectively)
as well as the decapeptide (SEQ ID NO: 3) previously shown to mimic
the biological action of native PSP94 were assessed in [H3]-Thymidine
uptake assay on PC-3 cells. Two separate experiments were conducted
with GMP-grade PCK3145.
As shown in the Figures 22 and 23, polypeptide PCK3145
exhibited a significant proliferation inhibition activity reflected
in the percentage of [H3]-Thymidine uptake. In the first experiment,
a reduction of nearly 40 % in [3H]-Thymidine uptake was observed at
PCK3145 concentration of 200 g/ml. In the second experiment,
although a two fold higher concentration of the PCK3145 was used
(i.e., 400 g/ml) only a 25 % inhibition was observed. Despite assay
to assay variation the overall degree of proliferative inhibitory
effect against PC-3 cell was markedly evident with the GMP grade
material. Treatment of PC-3 cells with the native PSP94 used as a
positive reference standard, exhibited a significant dose dependent
reduction in cell proliferation with almost 50 % reduction in the
[H3]-Thymidine uptake following 72 hours exposure (Figure 24).
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EXAMPLE 15
IN VITRO EFFECT OF PCK3145 ON PC-3 CELLS
(APOPTOSIS-Figure 25)
Apoptosis of PC-3 cells, following a 72 hours exposure to
PCK3145 at 500 g/ml concentration, was evaluated in supplemented
media by DNA fragmentation assay. Doxorubicin was used as a reference
positive control. Untreated cells and PCK3145-treated cells were
harvested and the DNA was isolated. Isolated DNA was run on a 1.2 %
agarose gel containing Ethidium Bromide (EtBr). As shown in Figure 25
treatment of PC-3 cells with polypeptide PKC3145 resulted in DNA
fragmentation evidenced by the ladder formation seen for fragmented
DNA. Lane 1 of the gel illustrated in figure 25 represents the DNA
marker (100 base pair DNA ladder). Lane 2 of the gel illustrated in
figure 25 represents a control of untreated PC-3 cells. Lane 3 of the
gel illustrated in figure 25 represents DNA laddering effect observed
for cells treated with doxorubicin at a concentration of 2 g/ml.
Lane 4 of the gel illustrated in figure 25 represents DNA laddering
effect observed for cells treated with PCK3145 (SEQ ID NO: 5).
EXAMPLE 16
IN VIVO EXPERIMENTS USING HUMAN PC-3 PROSTATE CANCER CELL LINE
(Figures 26-27)
Studies PC3-6 and PC3-12 (Figures 26 - 27) are consecutive
group experiments designed to characterize the in vivo activity of
PCK3145 in the human PC-3 prostate cancer nude mouse xenograft model
and to explore relationships between dose, route and schedule of
administration and the efficacy parameters of tumor growth (volume).
PC-3 cells harvested in mid-log phase were inoculated at 5 x
106 cells per mice via the subcutaneous route in the mice's back
area. Tumors grown from this inoculum were excised at approximately
day 32 to 35 post-tumor implantation (p.t.i) when tumor volume
reached 200-300 mm3 (i.e., cu mm). The necrotic tissue was removed
and the viable tumor mass cut into small pieces (approximately 1 to 3
mm3) were implanted SC in the flank region at two opposite sites of
the mouse. Treatment with various concentrations of PCK3145 was
initiated at day 3 post-tumor implantation (p.t.i) and was continued
daily for 21 days. Subcutaneous injections were done below tumor
growth sites. Intra-peritoneal injections were performed in the
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abdominal region. Intra-venous injections were performed via the
lateral tail vein. The experiment was terminated 24 hours after the
last treatment. Tumor measurements were taken at Days 11, 14, 16, 18,
20, 22 and 24 post-tumor implantation (p.t.i) . Tumor volumes were
calculated according to formula (axb2 x 0.5), where a - is the length
of the long diameter, and b-is the width of the perpendicular small
diameter.
Study No: PC3-6 illustrates the efficacy of PCK3145, injected
subcutaneously, in tumor growth retardation in Nude mice, which have
received PC-3 implants. Mice were separated in different group each
receiving various amounts of PCK3145 (SEQ ID NO: 5) or control
reagents. The different groups used in these experiments are
illustrated in table 6 below. Each group contained 10 mice.
Doxorubicin was administered as single bolus intra-venous injection
on days 3 and 11 post-tumor implantation (p.t.i).
TABLE 6
Treatment Test and Dose Level No. of No. of
group control ( g/kg/day) animals tumors
articles
1 Negative PBS 0 10 20
control
2 Positive Doxorubicin 10000 10 20
control
3 PCK3145 0.1 10 20
4 PCK3145 1 10 20
5 PCK3145 10 10 20
Results of this study (Figure 26) demonstrated a significant
PC-3 tumor growth retardation following treatment with PCK3145 at 10
g/kg/day. This anti-tumor effect was evidenced by a statistically
significant decrease in percentage of tumor growth observed at days
11, 14, 16, 18 , 21 and 24 after tumor implantation with respective
p-values ranging from p=0.001 to 0.002, in comparison to the control
PBS-treated group (p values measured by unpaired Student's t-test at
p<0.05 as cut-off limit) . Doxorubicin, a potent chemotherapeutic
agent, was used as reference gold standard and demonstrated a highly
significant anti-tumor therapeutic effect. ANOVA analysis of
variance, Dunnett's test, Kruskal-Wallis and Dunn's test analysis of
data confirmed statistical significance of the observed anti-tumor
effect.
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Study No: PC3-12 illustrates the efficacy of PCK3145 in tumor
growth retardation in Nude mice, which have received PC-3 implants.
Mice were separated in different group each receiving various amounts
of PCK3145 (SEQ ID NO: 5) or control reagents. PCK3145 was injected
either through intra-venous or intra-peritoneal route. The different
groups used in these experiments are illustrated in table 7 below.
Each group contained 9 mice.
TABLE 7
Treatment Test and Dose level No. of No. of
groups control ( g/kg/day) animals tumors
articles
1 Negative PBS 0 9 18
control
2 PCK3145 IV 10 9 18
3 PCK3145 IV 100 9 18
4 PCK3145 IV 500 9 18
5 PCK3145 IV 1000 9 18
6 PCK3145 IP 100 9 18
7 PCK3145 IP 1000 9 18
Results of this experiment (Figure 27) demonstrated a
significant tumor growth retardation following treatment with PCK3145
at 100 pg/kg/day via the intra-venous route. This effect was
statistically significant at days 13, 17 and 20 after tumor
implantation when compared by Student's t-test (p-values were
p=0.005, 0.025 and 0.011, respectively for each time-point) (p values
measured by unpaired Student's t-test at p<0.05 as cut-off limit). No
significant anti-tumor effect was observed following PCK3145
treatment at the other dosage levels of 10, 500 and 1000 .tg/kg/day
injected via the intra-venous route. However a trend towards
significance was observed following treatment with 500 and
1000 g/kg/day doses of PCK3145. Treatment of mice with PCK3145 at
100 and 1000 pg/kg/day administered via the intra-peritoneal route
showed a similar tumor growth retardation trend with statistically
less significant difference observed at day 13 p.t.i (p=0.056) (p
values measured by unpaired Student's t-test at p<0.05 as cut-off
limit) at the highest dose of 1000 g/kg/day (Figure 27).
During the course of experimentation using the human PC-3
prostate cancer nude mouse xenograft model, results obtained have
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suggested that subcutaneous PCK3145 injection of mice at a site
(i.e., scruff of the neck) distant from tumor site, might not be
efficacious enough and will unlikely may unlikely result in an anti-
tumor effect, at least in the experimental conditions tested (doses
of PCK3145 tested: 10 pg/kg/day and 100 pg/kg/day). The use of the
scruff of the neck as a subcutaneous injection site represents an
optimal site for immune response induction rather than a route for
therapeutic product administration and as such, selection of this
site is expected to be a sub-optimal site for tumor efficacy
evaluation.
EXAMPLE 17
IN VIVO EXPERIMENTS USING DUNNING RAT MAT LY LU PROSTATE CANCER
LINE
(Figures 28-30)
Anti-tumor efficacy evaluation of PCK3145 against Mat Ly Lu
(MLL) tumor implanted in Copenhagen rats was performed. (Protocol
based on S. Garde et al.; The Prostate, 22: 225-233, 1993). Mat LyLu
tumor cells were harvested in mid-log phase from the culture flasks
by trypsinization, were centrifuged at 1200 rotation per minute (rpm)
and washed three timed with Hanks balanced salt solution (HBSS).
Following washing, cells were counted and adjusted to a concentration
of 5 x 106 cells/ml in HBSS. A 0.1 ml volume of tumor cell inoculum
containing 5 x 105 cells was administered subcutaneously into the
flank region of each rat. Treatment started at day 3 post-tumor
implantation (p.t.i) by local subcutaneous injection (i.e., in the
shaved back area just below tumor implantation site) of various
PCK3145 concentrations. This treatment was continued daily for 16
days. Experiments were terminated 24 hours after the last treatment.
Tumor measurements were taken at days 7, 9, 11, 14, 16 and 18. Tumor
volumes are calculated according to formula (axb2 x 0.5), where a -
is the length of the long diameter, b-width of the perpendicular
small diameter. At day 19 tumors of individual rats were excised and
weighed.
Study No: MLL-5 illustrates the efficacy of PCK3145 (SEQ ID NO:
5) compared with polypeptide 7-21 (SEQ ID NO: 4) and polypeptide 76-
94 (SEQ ID NO: 6) in tumor growth retardation in Copenhagen rats,
which have received Mat Ly Lu implants. Mice were separated in
different groups, each receiving various amount of PCK3145 (SEQ ID
NO: 5) or control reagents. PCK3145 was injected through the
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subcutaneous route. The different groups used in these experiments
are illustrated in table 8 below. Each group contained 8 mice.
TABLE 8
Treatment Test and Dose Level No. of No. of
groups control ( g/kg/day) animals tumors
articles
1 Negative PBS 0 8 8
control
2 Polypeptide 10 8 8
7-21
3 Polypeptide 1 8 8
7-21
4 PCK3145 10 8 8
5 PCK3145 1 8 8
6 Polypeptide 10 8 8
76-94
7 Polypeptide 1 8 8
76-94
Results of this study (Figure 28) demonstrated a significant
anti-tumor effect following administration of PCK3145 at
10 g/kg/day. This was evidenced by a significant tumor volume
reduction at days 11 (p=0.006), 13 (p=0.00001), 16 (p=0.002) and
18(p=0.004), post-tumor cell implantation compared to control PBS-
treated group (p values measured by unpaired Student's t-test at
p<0.05 as cut-off limit). No significant effect was detectable
following PCK3145 treatment at 1 g/kg/day. It was of interest to
note that the amino-terminus polypeptide 7-21 also demonstrated
comparable anti-tumor effect, which was also observed in the PC-3
nude mouse xenograft model, indicating the possibility of an
overlapping active site between the N-terminus and the central
regions of the PSP94 protein. This was evidenced by a significant
tumor volume reduction observed at day 13 (p=0.05), 16 (p=0.00005),
and 18 (p=0.01) in mice treated with polypeptide 7-21) (p values
measured by unpaired Student's t-test at p<0.05 as cut-off limit).
Study No: MLL-6 illustrates the efficacy of PCK3145 (SEQ ID NO:
5) in tumor growth retardation in Copenhagen rats, which have
received Mat Ly Lu implants. Mice were separated in different group
each receiving various amounts of PCK3145 (SEQ ID NO: 5) or control
reagents. PCK3145 was injected through the subcutaneous route. The
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different groups used in these experiments are illustrated in table 9
below. Each group contained 8 mice. Doxorubicin was administered as
single bolus via intra-venous injection on day 3 p.t.i.
TABLE 9
Treatment Test and Dose level No. of No. of
groups control ( g/kg/day) animals tumors
articles
1 (Negative PBS 0 8 8
control)
2 Doxorubicin 5000 8 8
3 PCK3145 10 8 8
4 PCK3145 100 8 8
5 Scrambled 10 8 8
polypeptide
6 Scrambled 100 8 8
polypeptide
Results of this study (Figures 29 and 30) demonstrated a
significant dose-dependent anti-tumor effect following administration
of PCK3145 at 10 and 100 g/kg/day. This was evidenced by a
significant tumor volume reduction (31 % over control) following
PCK3145 treatment especially with 100 g/kg/day at days 14, 16 and 18
post-tumor cell implantation (Figure 29). The p-value versus negative
control-treated group (i.e., scrambled polypeptide (PB111)) was
highly significant at p=0.0000062 (p values measured by unpaired
Student's t-test at p<0.05 as cut-off limit). A moderate extent of
growth retardation (marginal statistical significance at p=0.03
versus control PBS-treated group) was also observed following
treatment with scrambled polypeptide at a concentration of 100
gg/kg/day (Figure 29) (p values measured by unpaired Student's t-
test at p<0.05 as cut-off limit). Doxorubicin treatment was highly
significant resulting in over 80 % reduction in tumor volumes. This
anti-tumor effect of PCK3145 at 100 pg/kg/day was also reproduced
following analysis of the tumor weights data. As shown in figure 30,
(tumor weight data) a significant reduction in tumor weights
(p=0.0003) was observed on day 18 p.t.i (p values measured by
unpaired Student's t-test at p<0.05 as cut-off limit). This
represented a 34 % reduction in tumor mass, a 20 gram difference
between the control (56.6 g) and PCK3145-treated at 100 pg/kg/day
group (37.2 g). This difference in tumor weights was also
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statistically significant when it was compared to the tumor weights
of the control scrambled polypeptide-treated rats given the same dose
of 100 g/kg/day (p=0.003) (p values measured by unpaired Student's
t-test at p<0.05 as cut-off limit). Comparison of the scrambled
polypeptide treated tumor weights with that of control PBS-untreated
tumor weights was not statistically significant (p=0.06) (p values
measured by unpaired Student's t-test at p<0.05 as cut-off limit).
EXAMPLE 18
EFFICACY OF PCK3145 AND TAXOTERE COMBINATION TREATMENT
In order to test for the efficacy of combination treatment, in
tumor growth retardation, PCK3145 and taxotere (i.e., docetaxel) were
co-administered in Nude mice previously inoculated with PC-3 tumor
cells. Mice were separated in different groups each receiving
PCK3145 alone or PCK3145 in combination with taxotere (administered
by separate routes) or control reagent (i.e., PBS). In this
experiment, the combination treatment was initiated against
relatively large tumor burdens. Tumors were allowed to grow beyond
the 50 to 60 mm3 size at which PCK3145 treatment usually becomes
inefficient. PCK3145 was injected through intravenous route every
other day for 28 days starting from day 1 when 50 to 60 mm3 size
subcutaneous tumors were apparent. Taxotere was injected by intra-
peritoneal route at a sub-optimal concentration of 2 mg/kg on days 4
and 11 after subcutaneous tumors were evident. The different groups
used in this experiment are illustrated in table 10 below. Each
group contained 11 mice.
TABLE 10
Treatment Test and Dose level No. of No of tumors
groups control. ( g/kg) animals
articles
1. Negative PBS 0 11 11
control
2. Positive Taxotere 2000 11 11
control
3. PCK3145 100 11 11
4. PCK3145 + 100 + 11 11
taxotere 2000
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Results of this experiment (Figure 31) demonstrate a significant
tumor growth retardation following combination treatment of PCK3145 and
taxotere. This effect is statistically significant at days 19 and 22 post-
tumor cell inoculation when compared by Student's t-test (p=0.02 at day 19
and p=0.047 at day 22), (p-values are measured by unpaired Student's t-test
at p<0.05 as a cut-off limit) and was markedly better than taxotere
administered alone at the same dose of 2 mg/kg (suboptimal dose).
The citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of prior
invention.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding,
it is readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
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Ser Cys Tyr Phe Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg
1 5 10 15
Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp
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Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Ile Ser
40 45
Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp Asn Cys
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Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val Glu Lys
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35 Lys Asp Pro Lys Lys Thr Cys Ser Val Ser Glu Trp Ile Ile
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Glu Ala Glu Ala Tyr Val Glu Phe Ser Cys Tyr Phe Ile Pro Asn Glu
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Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Giy Asn
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Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys
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Thr Cys Tyr Glu Thr Glu Ile Ser Cys Cys Thr Leu Val Ser Thr Pro
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Val Gly Tyr Asp Lys Asp Asn Cys Gin Arg Ile Phe Lys Lys Glu Asp
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Cys Lys Tyr Ile Val Val Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser
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Val Ser Glu Trp Ile Ile
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ACTTGCTACG AAACAGAAAT TTCATGTTGC ACCCTTGTTT CTACACCTGT GGGTTATGAC 180
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Ile
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20 25
2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro
20 25
2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val
20 25
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CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly
25
2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr
20 25
2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp
20 25 30
2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
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CA 02359650 2001-10-15
(A) LENGTH: 31
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Giy Tyr Asp Lys
25 30
20 2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn
2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34
-82-
CA 02359650 2001-10-15
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys
2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln
40
2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg
35
2) INFORMATION FOR SEQ ID NO: 31:
-83-
CA 02359650 2001-10-15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys Gln Arg Ile
20
2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38
25 (B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
30 (vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
35 1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe
2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys
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CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gin Arg Ile Phe Lys Lys
35 40
2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu
35 40
2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
- 85 -
CA 02359650 2001-10-15
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp
35 40
2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys
40
2) INFORMATION FOR SEQ ID NO: 38:
35 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys
35 40
2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
-86-
CA 02359650 2001-10-15
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr
15 35 40 45
2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 46
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile
35 40 45
2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Iie Val
35 40 45
2) INFORMATION FOR SEQ ID NO: 42:
-87-
CA 02359650 2001-10-15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys Gin Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gin Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu
2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
-88-
CA 02359650 2001-10-15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys
10
2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51
15 (B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
20 (vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
25 Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
40 45
Glu Lys Lys
35 50
2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp
-89-
CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
20 35 40 45
Giu Lys Lys Asp Pro
25
2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54
(B) TYPE: AMINO ACID
30 (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
35 (A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys
50
2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
-90-
CA 02359650 2001-10-15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gin Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys
50 55
2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gin Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr
50 55
2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gin Arg Ile Phe Lys Lys Giu Asp Cys Lys Tyr Ile Val Val
35 40 45
-91-
CA 02359650 2001-10-15
Glu Lys Lys Asp Pro Lys Lys Thr Cys
50 55
2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser
50 55
2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val
50 55
2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60
(B) TYPE: AMINO ACID
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(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val Her
50 55 60
2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Her Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val Her Glu
55 60
50 2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
-93-
CA 02359650 2001-10-15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val Ser Glu Trp
50 55 60
2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 1(D 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val Ser Glu Trp Ile
50 55 60
2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Ile Ser Cys Cys Thr Leu Val Ser Thr Pro Val Gly Tyr Asp Lys Asp
20 25 30
-94-
CA 02359650 2001-10-15
Asn Cys Gln Arg Ile Phe Lys Lys Glu Asp Cys Lys Tyr Ile Val Val
35 40 45
Glu Lys Lys Asp Pro Lys Lys Thr Cys Ser Val Ser Glu Trp Ile Ile
50 55 60
2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
1 5 10 15
2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu
1 5 10 15
Thr
2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Ile Asn Ser Giu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr
1 5 10 15
Glu Thr
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2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys
1 5 10 15
Tyr Glu Thr
2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr
1 5 10 15
Cys Tyr Glu Thr
40
2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
45 (B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
50 (vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
55 Lys His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys
1 5 10 15
Thr Cys Tyr Glu Thr
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CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr
1 5 10 15
Cys Thr Cys Tyr Glu Thr
20
2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu
1 5 10 15
Thr Cys Thr Cys Tyr Glu Thr
20
2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Lys Giy Asn Lys His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys
1 5 1C) 15
Glu Thr Cys Thr Cys Tyr Giu Thr
20
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CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn
1 5 10 15
Cys Glu Thr Cys Thr Cys Tyr Glu Thr
25
2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp
1 5 10 15
Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25
2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr
1 5 10 15
Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25
-98-
CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln
1 5 10 15
Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
25
20 2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp
1 5 10 15
Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25
2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
Arg Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu
1 5 10 15
Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25 30
2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
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CA 02359650 2001-10-15
(A) LENGTH: 31
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Thr Arg Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser
1 5 10 15
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
25 30
2) INFORMATION FOR SEQ ID NO: 75:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Ser Thr Arg Lys Cys Met Asp Leu Lys G_ly Asn Lys His Pro Ile Asn
1 5 10 15
Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25 30
2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile
1 5 10 15
Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu
20 25 30
Thr
2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34
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CA 02359650 2001-10-15
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro
1 5 10 15
Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr
20 25 30
Glu Thr
2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly Asn Lys His
1 5 10 15
Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys
20 25 30
Tyr Glu Thr
40
2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly Asn Lys
1 5 10 15
His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr
20 25 30
Cys Tyr Glu Thr
35
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CA 02359650 2001-10-15
2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly Asn
1 5 10 15
Lys His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys
20 25 30
Thr Cys Tyr Glu Thr
25 2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
30 (D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys Gly
1 5 10 15
Asn Lys His Pro Ile Asn Ser Glu Trp Gin Thr Asp Asn Cys Glu Thr
20 25 30
Cys Thr Cys Tyr Glu Thr
45
2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39
50 (B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
55 (vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
60 Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu Lys
1 5 10 15
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CA 02359650 2001-10-15
Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn Cys Glu
20 25 30
Thr Cys Thr Cys Tyr Glu Thr
35
2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp Leu
1 5 10 15
Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn Cys
20 25 30
Glu Thr Cys Thr Cys Tyr Glu Thr
40
2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met Asp
1 5 10 15
Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp Asn
20 25 30
Cys Glu Thr Cys Thr Cys Tyr Glu Thr
35 40
2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
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CA 02359650 2001-10-15
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
Phe Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys Met
1 5 10 15
Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr Asp
20 25 30
Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr.
35 40
2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Tyr Phe Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys Cys
1 5 10 15
Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln Thr
20 25 30
Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
40
2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Cys Tyr Phe Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg Lys
1 5 10 15
Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp Gln
20 25 30
Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
35 40
2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
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CA 02359650 2001-10-15
(A) LENGTH: 45
(C) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
Ser Cys Tyr Phe Ile Pro Asn Glu Gly Val Pro Gly Asp Ser Thr Arg
1 5 10 15
Lys Cys Met Asp Leu Lys Gly Asn Lys His Pro Ile Asn Ser Glu Trp
25 30
Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
35 40 45
2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(ix) FEATURE
(A) NAME/KEY : Modified site
(B) LOCATION : 1
(D) OTHER INFORMATION : The residue in this
position is either glutamic acid, asparagine, or
aspartic acid.
(ix) FEATURE
(A) NAME/KEY : Modified site
(B) LOCATION : 4
(D)OTHER INFORMATION : The residue in this position
is either threonine, or serine.
(ix) FEATURE
(A) NAME/KEY : Modified site
(B) LOCATION : 6
(D)OTHER INFORMATION : The residue in this position
is either glutamic acid, asparagine, or aspartic
acid.
(ix) FEATURE
(A) NAME/KEY : Modified site
(B) LOCATION : 8
(D)OTHER INFORMATION : The residue in this position
is either glutamic acid, asparagine, or aspartic
acid.
(ix) FEATURE
(A)NAME/KEY : Modified site
(B)LOCATION : 9
(D)OTHER INFORMATION : The residue in this position
is either threonine, or serine.
(ix) FEATURE
(A)NAME/KEY : Modified site
(B)LOCATION : 11
(D)OTHER INFORMATION : The residue in this position
is either threonine, or serine.
(ix) FEATURE
(A)NAME/KEY : Modified site
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CA 02359650 2001-10-15
(B)LOCATION : 13
(D)OTHER INFORMATION : The residue in this position
is either tyrosine, or phenylalanine.
(ix) FEATURE :
(A)NAME/KEY : Modified site
(B)LOCATION : 14
(D)OTHER INFORMATION : The residue in this position
is either glutamic acid, asparagine, or aspartic
acid.
(ix) FEATURE
(A)NAME/KEY : Modified site
(B)LOCATION : 15
(D)OTHER INFORMATION : The residue in this position
is either threonine, or serine.
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Xaa Trp Gln Xaa Asp Xaa Cys Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
1 5 10 15
2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Glu Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Trp Gln Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
20 25 30
2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
- 106 -
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Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Trp
20 25 30
Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
35 40 45
2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULE TYPE:
(vi)ORIGINAL SOURCE:
(A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Glu Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu
1 5 10 15
Trp Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Trp
20 25 30
Gin Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Trp Gin
40 45
Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr
50 55 60
- 107 -
