U.S. patent application number 12/879575 was filed with the patent office on 2011-01-13 for q3 sparc deletion mutant and uses thereof.
This patent application is currently assigned to Abraxis BioScience, LLC. Invention is credited to Neil P. Desai, Patrick Soon-Shiong, Vuong Trieu.
Application Number | 20110009337 12/879575 |
Document ID | / |
Family ID | 37000156 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110009337 |
Kind Code |
A1 |
Trieu; Vuong ; et
al. |
January 13, 2011 |
Q3 SPARC DELETION MUTANT AND USES THEREOF
Abstract
The invention provides for SPARC polypeptides with a mutation
corresponding to a deletion of the third glutamine in the mature
fault of the human SPARC protein, nucleic acids encoding such
polypeptides, antibodies against such polypeptides, and methods of
the use of such polypeptides, nucleic acids, and antibodies.
Inventors: |
Trieu; Vuong; (Calabasas,
CA) ; Desai; Neil P.; (Los Angeles, CA) ;
Soon-Shiong; Patrick; (Los Angeles, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
Abraxis BioScience, LLC
Los Angeles
CA
|
Family ID: |
37000156 |
Appl. No.: |
12/879575 |
Filed: |
September 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11964390 |
Dec 26, 2007 |
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12879575 |
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11356829 |
Feb 17, 2006 |
7332568 |
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11964390 |
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60654261 |
Feb 18, 2005 |
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Current U.S.
Class: |
514/19.5 ;
514/19.3; 514/21.2 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/191 20130101; C07K 14/4747 20130101; G01N 2800/52 20130101;
C07K 16/18 20130101; A61P 35/00 20180101; C07K 14/82 20130101; C07K
14/47 20130101; C07K 14/51 20130101; A61P 35/02 20180101 |
Class at
Publication: |
514/19.5 ;
514/19.3; 514/21.2 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 35/00 20060101 A61P035/00 |
Claims
1-64. (canceled)
65. A method of treating a disease in a mammal comprising
administering a SPARC polypeptide comprising SEQ ID NO: 1 to the
mammal.
66. The method of claim 65, further comprising administering one or
more additional therapeutic agents.
67. The method of claim 66, wherein the one or more additional
therapeutic agents are selected from the group consisting of
therapeutic radionuclides, adriamycin, ansamycin, antibiotics,
asparaginase, bleomycin, busulphan, cisplatin, carboplatin,
carmustine, capecitabine, chlorambucil, cytarabine,
cyclophosphamide, camptothecin, dacarbazine, dactinomycin,
daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,
epothilones, floxuridine, fludarabine, 5-fluorouracil, gemcitabine,
hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,
mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin
and derivatives, mitomycin, mitotane, mitoxantrone, nitrosurea,
paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine,
rituximab, streptozocin, teniposide, thioguanine, thiotepa,
taxanes, vinblastine, vincristine, vinorelbine, taxol,
combretastatins, discodermolides, transplatinum, anti-vascular
endothelial growth factor compounds, anti-epidermal growth factor
receptor compounds, tyrosine kinase inhibitors, and biologically
active agents.
68. The method of claim 67, wherein the additional therapeutic
agent is 5-fluorouracil.
69. The method of claim 66, wherein the additional therapeutic
agent is comprised of paclitaxel albumin nanoparticles.
70. The method of claim 65, further comprising the administration
of one or more angiogenesis inhibitors.
71. The method of claim 65, wherein the disease is a cell
proliferative disease.
72. The method of claim 71, wherein the cell proliferative disease
is a cancer.
73. The method of claim 72, wherein the cancer is selected from the
group consisting cancer of the brain, oral cavity and pharynx,
esophagus, stomach, small intestine, colon, rectum, anus, liver,
gall bladder, pancreas, larynx, lung, bronchus, uterine cervix,
uterine corpus, ovary vulva, vagina, prostate, testis, and penis,
genital systems, urinary bladder, kidney, renal pelvis, ureter,
sarcomas, meningioma, Hodgkin's disease, Non-Hodgkin's lymphoma,
multiple myeloma, and leukemia.
74. The method of claim 65, wherein the mammal is a human.
75. A method of treating a disease in a mammal comprising
administering a SPARC polypeptide comprising SEQ ID NO: 2 to the
mammal.
76. The method of claim 75, further comprising administering one or
more additional therapeutic agents.
77. The method of claim 76, wherein the one or more additional
therapeutic agents are selected from the group consisting of
therapeutic radionuclides, adriamycin, ansamycin, antibiotics,
asparaginase, bleomycin, busulphan, cisplatin, carboplatin,
carmustine, capecitabine, chlorambucil, cytarabine,
cyclophosphamide, camptothecin, dacarbazine, dactinomycin,
daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,
epothilones, floxuridine, fludarabine, 5-fluorouracil, gemcitabine,
hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,
mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin
and derivatives, mitomycin, mitotane, mitoxantrone, nitrosurea,
paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine,
rituximab, streptozocin, teniposide, thioguanine, thiotepa,
taxanes, vinblastine, vincristine, vinorelbine, taxol,
combretastatins, discodermolides, transplatinum, anti-vascular
endothelial growth factor compounds, anti-epidermal growth factor
receptor compounds, tyrosine kinase inhibitors, and biologically
active agents.
78. The method of claim 77, wherein the additional therapeutic
agent is 5-fluorouracil.
79. The method of claim 76, wherein the additional therapeutic
agent is comprised of paclitaxel albumin nanoparticles.
80. The method of claim 75, further comprising the administration
of one or more angiogenesis inhibitors.
81. The method of claim 75, wherein the disease is a cell
proliferative disease.
82. The method of claim 81, wherein the cell proliferative disease
is a cancer.
83. The method of claim 82, wherein the cancer is selected from the
group consisting cancer of the brain, oral cavity and pharynx,
esophagus, stomach, small intestine, colon, rectum, anus, liver,
gall bladder, pancreas, larynx, lung, bronchus, uterine cervix,
uterine corpus, ovary vulva, vagina, prostate, testis, and penis,
genital systems, urinary bladder, kidney, renal pelvis, ureter,
sarcomas, meningioma, Hodgkin's disease, Non-Hodgkin's lymphoma,
multiple myeloma, and leukemia.
84. The method of claim 75, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of copending U.S.
patent application Ser. No. 11/356,829, filed Feb. 17, 2006, which
is hereby incorporated by reference in its entirety. This patent
application claims the benefit of U.S. Provisional Patent
Application No. 60/654,261 filed on Feb. 18, 2005, which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for treating cancer, other
diseases involving abnormal proliferative, hyperplastic,
remodeling, and inflammatory activity in tissues and organs using
antibody or other suitable ligands recognizing SPARC. The invention
also relates to mutant SPARC polypeptides and nucleic acids and
methods of their use, as well as methods for targeting, methods for
imaging, and methods for determining the response of mammalian
tumors to anti-SPARC therapy.
BACKGROUND OF THE INVENTION
[0003] Secreted protein acidic and rich in cysteine (also known as
osteonectin, BM40, or SPARC) (hereafter "SPARC"), is a
matrix-associated protein that elicits changes in cell shape,
inhibits cell-cycle progression, and influences the synthesis of
extracellular matrix (Bradshaw et al., Proc. Nat. Acad. Sci. USA
100: 6045-6050 (2003)). The murine SPARC gene was cloned in 1986
(Mason et al., EMBO J. 5: 1465-1472 (1986)) and a full-length human
SPARC cDNA was cloned and sequenced in 1987 (Swaroop et al.,
Genomics 2: 37-47 (1988)). SPARC expression is developmentally
regulated, and is predominantly expressed in tissues undergoing
remodeling during normal development or in response to injury. For
example, high levels of SPARC protein are expressed in developing
bones and teeth (see, e.g., Lane et al., FASEB J., 8, 163 173
(1994); Yan & Sage, J. Histochem. Cytochem. 47:1495-1505
(1999)).
[0004] SPARC is upregulated in several aggressive cancers, but is
absent in the corresponding inormal tissues (Porter et al., J.
Histochem. Cytochem., 43, 791 (1995)). SPARC expression is induced
among a variety of tumors (e.g., bladder, liver, ovary, kidney,
gut, and breast). In bladder cancer, for example, SPARC expression
has been associated with advanced carcinoma. Invasive bladder
tumors of stage T2 or greater have been shown to express higher
levels of SPARC relative to bladder tumors of stage T1 (or less
superficial tumors), and poorer prognosis (see, e.g., Yamanaka et
al., J. Urology, 166, 2495 2499 (2001)). In meningiomas, SPARC
expression has been associated only with invasive tumors (see,
e.g., Rempel et al., Clincal Cancer Res., 5, 237 241 (1999)). SPARC
expression also has been detected in 74.5% of in situ invasive
breast carcinoma lesions (see, e.g., Bellahcene, et al., Am. J.
Pathol., 146, 95 100 (1995)), and 54.2% of infiltrating ductal
carcinoma of the breast (see, e.g., Kim et al., J. Korean Med.
Sci., 13, 652 657 (1998)). SPARC expression also has been
associated with frequent microcalcification in breast cancer (see,
e.g., Bellahcene et al., supra), suggesting that SPARC expression
may be responsible for the affinity of breast metastases for the
bone.
[0005] Surprisingly, SPARC has also been shown to have anti-tumor
activity in some systems. SPARC is a potent cell cycle inhibitor
that arrests cells in mid-G.sub.1 (Yan & Sage, J. Histochem.
Cytochem. 47:1495-1505 (1999)) and the inducible expression of
SPARC has been shown to inhibit breast cancer cell proliferation in
an in vitro model system (Dhanesuan et al., Breast Cancer Res.
Treat. 75:73-85 (2002)). Similarly, exogenous SPARC can reduce the
proliferation of both HOSE (human ovarian surface epithelial) and
ovarian cancer cells in a concentration-dependent manner. In
addition, SPARC induces apoptosis in ovarian cancer cells. Further
evidence for SPARC receptors present on cells such as ovarian
epithelial cells has been report. It has been proposed that the
binding of SPARC to its receptor is likely to trigger
tissue-specific signaling pathways that mediate its tumor
suppressing functions (Yiu et al., Am. J. Pathol. 159:609-622
(2001)). Purified SPARC has also been reported to potently inhibit
angiogenesis and significantly impair neuroblastoma tumor growth in
an in vivo xenograft model system (Chlenski et al., Cancer Res.
62:7357-7363 (2002)).
[0006] SPARC also plays a role in non-neoplastic proliferative
diseases. Mesangial cell proliferation is a characteristic feature
of many glomerular diseases and often precedes extracellular matrix
expansion and glomerulosclerosis. In a model of experimental
mesangioproliferative glomerulonephritis, SPARC mRNA was increased
5-fold by day 7 and was identified in the mesangium by in situ
hybridization. However, recombinant SPARC or a synthetic SPARC
peptide inhibited platelet-derived-growth-factor-induced mesangial
cell DNA synthesis in vitro (Pichler et al., Am. J. Pathol.
148(4):1153-67 (1996)). Similarly, while renal enlargement, due to
hyperplasia, hypertrophy, and increase inter-cellular matrix, is a
characteristic feature of diabetes in humans, kidney SPARC mRNA
levels fell in diabetic animals. In addition, the onset of
diabetes-related kidney growth is associated with a reduction in
SPARC mRNA and protein (Gilbert et al., Kidney Int. 48(4):1216-25
(1995)).
[0007] SPARC has been implicated in the pathogenesis of
atherosclerotic lesions. Plasma SPARC levels are elevated in
patients with coronary artery disease (Masahiko et al., Obesity
Res. 9:388-393 (2001)). The proliferation of vascular smooth muscle
cells in the arterial intima plays a central role in the
pathogenesis of atherosclerosis. SPARC is expressed in vascular
smooth muscle cells and macrophages associated with atherosclerotic
lesions. In addition, SPARC has been hypothesized to regulate the
action of platelet-derived growth factor during vascular injury
(Masahiko et al., Obesity Res. 9:388-393 (2001); Raines et al.,
Proc. Natl. Acad. Sci. USA 89:1281-1285 (1992)). A stimulating
effect of SPARC on endothelial PAI-1 production has been reported
at the site of vascular injury (Hasselaar et al., J. Biol. Chem.
266:13178-13184 (1991)) and has been postulated to accelerate
atherosclerosis (Masahiko et al., Obesity Res. 9:388-393
(2001)).
[0008] SPARC has affinity for a wide variety of ligands including
cations (e.g., Ca 2+, Cu 2+, Fe 2+), growth factors (e.g., platelet
derived growth factor (PDGF), and vascular endothelial growth
factor (VEGF)), extracellular matrix (ECM) proteins (e.g., collagen
I V and collagen IX, vitronectin, and thrombospondin 1),
endothelial cells, platelets, albumin, and hydroxyapaptite (see,
e.g., Lane et al., FASEB J., 8, 163 173 (1.994); Yan & Sage, J.
Histochem. Cytochem. 47:1495-1505 (1999)). SPARC is also known to
bind albumin (see, e.g., Schnitzer, J. Biol. Chem., 269, 6072
(1994)).
[0009] Antibody therapy is an effective method for controlling
disease wherein a specific protein marker can be identified.
Examples include Avastin (anti-VEGF antibody), Rituxan (anti-CD20
antibody), and Remicade (anti-TNF antibody). As such, antibody
against SPARC represent an important therapeutic agent for treating
human and other mammalian tumors, or other proliferative,
hyperplastic, remodeling, and inflammatory disorders, that express
the SPARC protein.
[0010] Accordingly, there is a need for novel forms of SPARC and
antibodies reactive with such novel forms of SPARC. The present
invention provides for such novel SPARC polypeptides, nucleic acids
which encode such novel SPARC polypeptides, and methods of use of
such novel SPARC polypeptides and nucleic acids. The invention
additionally provides for antibodies against SPARC
polypeptides.
BRIEF SUMMARY OF THE INVENTION
[0011] The glutamine corresponding to amino acid 20 in SEQ ID NO: 1
(the sequence of the unprocessed primary translation product)
corresponds to the glutamine at amino acid position 3 in the mature
protein (the polypeptide without the 17 amino acid SPARC leader
sequence) (the "Q3" glutamine). The invention provides for an
isolated SPARC polypeptide comprising an amino acid sequence
wherein the glutamine corresponding to amino acid 20 in SEQ ID NO:
1 is deleted (hereafter "Q3 SPARC deletion mutant polypeptide"). In
particular, the invention provides for an isolated human Q3 SPARC
deletion mutant polypeptide. The invention also provides for an
isolated SPARC polypeptide, wherein the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, which corresponds to a mature
Q3 SPARC deletion mutant polypeptide. The invention further
provides for an isolated nucleic acid molecule encoding a Q3 SPARC
deletion mutant polypeptide. In particular, the invention provides
for an isolated nucleic acid molecule encoding human Q3 SPARC
deletion mutant polypeptide. Isolated nucleic acids encompassed by
the invention include, but are not limited to, nucleic acids
comprising the sequence of SEQ ID NO: 3.
[0012] The invention also provides for a vector comprising a
nucleic acid molecule encoding a Q3 SPARC deletion mutant
polypeptide including, but not limited to, wherein the vector
further comprises a promoter controlling the expression of the Q3
SPARC deletion mutant polypeptide encoding nucleic acid sequences.
In addition, the invention provides for a cell comprising a nucleic
acid molecule encoding a Q3 SPARC deletion mutant polypeptide,
wherein the cell is either a prokaryotic cell or a eukaryotic cell.
The invention further provides for a method of making a Q3 SPARC
deletion mutant polypeptide of comprising: (a) transforming cells
with a nucleic acid encoding a Q3 SPARC deletion mutant
polypeptide; (b) inducing the expression of the Q3 SPARC deletion
mutant polypeptide by the transformed cells, and (c) purifying the
Q3 SPARC deletion mutant polypeptide.
[0013] In another embodiment the invention provides for composition
comprising a Q3 SPARC deletion mutant polypeptide or a nucleic acid
encoding a Q3 SPARC deletion mutant polypeptide and a
pharmaceutically acceptable carrier and method of treating a
disease comprising administering the Q3 SPARC deletion mutant
polypeptide and carrier. Such a method can be used in accordance
with the invention to treat a disease including, without
limitation, wherein the disease is a cell proliferative disease
(e.g., cancer, benign tumor, atherosclerosis, vascular restenosis).
The invention also provides for a method of sensitizing a disease
comprising administering a Q3 SPARC deletion mutant polypeptide
including wherein the disease is a cell proliferative disease
(e.g., cancer, benign tumor, atherosclerosis, vascular restenosis).
In addition, the invention provides for compositions comprising a
Q3 SPARC deletion mutant polypeptide, wherein the polypeptide is
coupled or conjugated to a therapeutic or diagnostic agent, such
as, e.g., a radioisotope or radioinuclide, drug, polypeptide or
toxin. Alternatively, the Q3 SPARC deletion mutant polypeptide can
be coupled or conjugated to molecule which stabilizes the Q3 SPARC
deletion mutant polypeptide in vivo, such as, e.g., a polyethylene
glycol. The Q3 SPARC deletion mutant polypeptide or nucleic acid
encoding a Q3 SPARC deletion mutant polypeptide and
pharmaceutically acceptable carrier can be administered through any
suitable route including, without limitation, intravenous,
intraperitoneal, intratumoral, or inhalational.
[0014] In one embodiment the invention provides for antibody or
fragment thereof with recognition for a SPARC polypeptide, in
particular for a Q3 SPARC deletion mutant polypeptide, and a
pharmaceutically acceptable carrier. Such an antibody with
recognition for a SPARC polypeptide, can be used in accordance with
the invention to, e.g., mediate complement activation and/or cell
mediated cytotoxicity against the tumor or other proliferative
disease.
[0015] In yet another embodiment, the invention provides for a
composition comprising a therapeutic agent coupled to an antibody
or fragment thereof with recognition for a SPARC polypeptide, in
particular for a Q3 SPARC deletion mutant polypeptide, and a
pharmaceutically acceptable carrier including, without limitation,
wherein said antibody or fragments thereof are humanized and
include monovalent Fab', divalent Fab2, scfv, diabody, or a chimera
(hereafter collectively an "anti-Q3 SPARC deletion mutant
antibody.") The antibody prepared in accordance with the invention
can be monoclonal or polyclonal, manufactured in non-human animals,
and be humanized. In addition, the invention provides for an
anti-SPARC antibody, in particular, an anti-Q3 SPARC deletion
mutant antibody, coupled or conjugated to a therapeutic agent,
wherein the therapeutic agent is a chemotherapeutic drug,
radionuclide, or peptide. Suitable therapeutic agents also include
therapeutic agents that are biological molecules such as, e.g, tTF
and TNF.
[0016] The Q3 SPARC deletion mutant polypeptide or anti-SPARC
antibody, such as, e.g., anti-Q3 SPARC deletion mutant antibody,
and pharmaceutically acceptable carrier can be administered through
any suitable route including, without limitation, intravenous,
intraperitoneal, intratumoral, or inhalational. Accordingly, the
invention provides for a method for delivering a therapeutic agent
to a disease site, such as, e.g., a tumor in a mammal, which method
comprises administering to the mammal a therapeutically effective
amount of a pharmaceutical composition, wherein the therapeutic
agent comprises a chemotherapeutic agent or radioactive agent
coupled to an pharmaceutically acceptable carrier and a
pharmaceutically acceptable carrier. Suitable tumors that can be
treated in accordance with the invention include, without
limitation, tumors located in the bladder, liver, ovary, kidney,
gut, brain, or breast of a human or non-human animal.
[0017] The invention additionally provides for a method for
delivering a diagnostic agent to a disease site, such as, e.g., a
tumor in a mammal, which method comprises administering to the
mammal a diagnostically effective amount of a pharmaceutical
composition. Suitable such compositions include, without
limitation, wherein the composition comprises a diagnostic agent
coupled to an anti-SPARC antibody, such as, e.g., an anti-Q3 SPARC
deletion mutant antibody, and a pharmaceutically acceptable
carrier. Suitable diagnostic agents include, without limitation,
radioactive agents, MRI contrast agents, X-Ray contrast agents,
ultrasound contrast agents, and PET contrast agents.
[0018] The invention provides for a method of classifying a disease
comprising detecting a SPARC polypeptide with a deletion of the
glutamine corresponding to amino acid position 20 in SEQ ID NO: 1
(i.e., detecting a Q3 SPARC deletion mutant polypeptide) including,
without limitation, wherein the disease is characterized by
cellular proliferation such as, e.g., cancer, benign tumor,
atherosclerosis or vascular restenosis. Suitable diseases also
include, without limitation, those where neoangiogenesis occurs. As
such, the invention further provides for a method of detecting a
SPARC polypeptide wherein the detected polypeptide comprises the
amino acid sequence of SEQ ID NO: 2.
[0019] The invention additionally provides for a method of
classifying a disease comprising detecting a nucleic acid encoding
a Q3 SPARC deletion mutant polypeptide, wherein the nucleic acid is
DNA or RNA. Further, the invention provides a method of classifying
a disease comprising detecting a nucleic acid, wherein the nucleic
acid detected comprises the nucleic sequence of SEQ ID NO: 3.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0020] FIG. 1 illustrates albumin and SPARC staining in MX 1 tumor
xenografts.
[0021] FIG. 2 illustrates transcytosis of paclitaxel across
endothelial cell monolayers.
[0022] FIG. 3 illustrates the sequencing strategy used to determine
the SPARC DNA sequence of the SPARC cDNA clone isolated from the
human prostate cDNA library.
[0023] FIG. 4A-U disclose the results of the sequencing reactions
depicted in FIG. 3.
[0024] FIG. 5 discloses the DNA sequence of SEQ ID NO: 3.
[0025] FIG. 6 discloses the amino acid sequence of SEQ ID NO: 4
which is a translation of SEQ ID NO: 5.
[0026] FIG. 7 discloses the amino acid sequence of SEQ ID NO: 2
which is a translation of SEQ ID NO: 6.
[0027] FIG. 8A-C illustrate a DNA sequence alignment of SEQ ID NO:
3 (Query) with the wild type SPARC cDNA sequence (Sbjct).
[0028] FIG. 9 illustrates an amino acid sequence alignment of the
wild type SPARC amino acid sequence (seq1) with SEQ ID NO: 4
(seq2).
[0029] FIG. 10A-B disclose a phylogenetic comparison of SPARC amino
acid sequences.
[0030] FIG. 11 depicts the gel electrophoresis results of
confirmatory restriction digests of the pVT1000Q3 plasmid.
[0031] FIG. 12 depicts the restriction map of the pVT1000Q3
plasmid.
[0032] FIG. 13 illustrates a mutagenic primer used in a site
directed mutagenesis method and the sequences targeted.
[0033] FIG. 14 illustrates the positions and sequences of PCR
primers used to detect the presence of nucleic acids encoding a Q3
SPARC deletion mutant polypeptide and/or a wild type SPARC
polypeptide ("Query", mutant sequence; "Sbjct", wild type
sequence).
[0034] FIG. 15 depicts the gel electrophoresis results of PCR
reactions used to detect the presence of nucleic acids encoding a
Q3 SPARC deletion mutant polypeptide and/or a wild type SPARC
polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The human SPARC gene encodes a 303 amino acid SPARC protein,
while mature SPARC is a 285 amino acid glycoprotein. After cleavage
of the signal sequence a 32-kD secreted form is produced which
migrates at 43 kD on SDS-PAGE because of glycosylation.
Crystallographic data indicates the SPARC protein has three modular
domains. The N-terminal domain is acidic and binds calcium. The
central "follistatin-like domain" contains amino acids involved in
the inhibition of angiogenesis and focal adhesion plaque formation
and the (K)GHK angiogenic peptide. The carboxyl terminal "E-C
domain" contains the amino acids involved in high affinity calcium
binding and the inhibition of cell spreading (Yan & Sage, J.
Histochem. Cytochem. 7:1495-1505, 1999).
[0036] The N-terminal SPARC domain (residues 1-52 after a
17-amino-acid signal sequence) is an acidic region rich in Aspartic
Acid and Glutamic Acid. The N-terminal domain binds several calcium
ions with low affinity, interacts with hydroxyapatite, and has been
postulated to play a role in bone mineralization. This N-terminal
SPARC domain has also been shown to inhibit cell spreading and
chemotaxis (Yan & Sage, J. Histochem. Cytochem. 47:1495-1505
(1999)). Further, the N-terminal domain contains the major
immunological epitopes of SPARC. However, this domain is the
divergent sequence among the family of SPARC-like proteins and
antibodies against SPARC have not been found to crossreact with or
recognize SPARC-like proteins (Yan & Sage, J. Histochem.
Cytochem. 7:1495-1505, 1999).
[0037] Two adjacent glutamines within the N-terminal domain at
amino acid positions 3 and 4 in the mature SPARC protein (amino
acids positions 20 and 21 in the unprocessed protein) are amine
acceptor sites which are the primary targets for crosslinking by
transglutaminas.sub.c. (Glutamine is represented by "Q" in the
single-letter amino acid code, thus, these amino acids are the "Q3"
and "Q4" amino acids in the mature SPARC protein.)
Transglutaminases catalyze the Ca-dependent transfer reaction
between the gamma-carboxamide group of a peptide-bound glutamine
residue and various primary amines. It has been proposed that
tissue-specific posttranslational modifications, such as
transglutaminase cross-linking, may modulate some of SPARC's
biological functions (Hohenadl et al., J. Biol. Chem.
270:23415-23420 (1995)).
[0038] The invention provides for an isolated SPARC polypeptide
comprising an amino acid sequence wherein the glutamine
corresponding to amino acid 20 in SEQ ID NO: 1 is deleted (a Q3
SPARC deletion mutant polypeptide). In particular, the invention
provides for an isolated human Q3 SPARC deletion mutant
polypeptide. The invention also provides for an isolated SPARC
polypeptide, wherein the polypeptide comprises the amino acid
sequence of SEQ ID NO: 2, which corresponds to a mature Q3 SPARC
deletion mutant polypeptide (the polypeptide without the 17 amino
acid SPARC leader sequence).
[0039] The invention further provides for an isolated nucleic acid
molecule encoding a Q3 SPARC deletion mutant polypeptide. In
particular, the invention provides for an isolated nucleic acid
molecule encoding human Q3 SPARC deletion mutant polypeptide.
Isolated nucleic acids encompassed by the invention include, but
are not limited to, nucleic acids comprising the sequence of SEQ ID
NO: 3. Accordingly, the invention provides for an isolated SPARC
polypeptide, wherein the polypeptide comprises the amino acid
sequence of SEQ ID NO: 4, which corresponds to the translation of
the SEQ ID NO: 3 DNA sequence from nucleotide 87 through nucleotide
992 and which results in a full length Q3 SPARC deletion mutant
polypeptide (a SPARC polypeptide with the 17 amino acid SPARC
leader sequence). Also accordingly, the invention provides for
isolated nucleic acids with the DNA sequences of SEQ ID NO: 5 and
SEQ ID NO: 6 which, respectively, encode polypeptides with the
amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 2. The DNA
sequence of SEQ ID NO: 6 represents nucleotides 138 through 992 of
SEQ ID NO: 3.
[0040] The invention provides for an isolated SPARC polypeptide
comprising an amino acid sequence wherein the glutamine
corresponding to amino acid 20 in SEQ ID NO: 1 is deleted.
Accordingly, the invention provides for an isolated Q3 SPARC
deletion mutant polypeptide and wherein the polypeptide is at least
about 10 amino acids long, preferably is at least about 15 amino
acids long, more preferably is at least about 20 amino acids long,
more preferably is at least about 30 amino acids long, more
preferably is at least about 40 amino acids long, more preferably
is at least about 50 amino acids long, even more preferably is at
least about 100 amino acids long. Further, the invention provides
for an isolated Q3 SPARC deletion mutant polypeptide comprising a
polypeptide wherein the sequences other than the glutamine
corresponding to amino acid 20 in SEQ ID NO: 1 are at least about
80% homologous to the corresponding sequences of SEQ ID NO: 1,
preferably at least about 90% homologous to the corresponding
sequences of SEQ ID NO: 1, even more preferably at least about 95%
homologous to the corresponding sequences of SEQ ID NO: 1, even
more preferably at least about 99% homologous to the corresponding
sequences of SEQ ID NO: 1. By "corresponding sequences of SEQ ID
NO: 1" it is meant, those sequences which align with the sequences
of SEQ ID NO: 1 wherein the region of alignment is at least about
10 amino acids long, preferably is at least about 15 amino acids
long, more preferably is at least about 20 amino acids long, more
preferably is at least about 30 amino acids long, more preferably
is at least about 40 amino acids long, more preferably is at least
about 50 amino acids long, even more preferably is at least about
100 amino acids long. Various methods of sequence alignment are
known in the biotechnology arts (see, e.g., Rosenberg, BMC
Bioinformatics 6:278 (2005); Altschul et al., FEBS J. 272(20):
5101-5109 (2005)).
[0041] The invention also provides for an isolated SPARC
polypeptide, wherein the polypeptide comprises the amino acid
sequence of SEQ ID NO: 2, including polypeptides comprising the
amino acid sequence of SEQ ID NO: 2 without any additional amino
acids added to the amino or carboxyl terminus, as well as
polypeptides comprising the amino acid sequence of SEQ ID NO: 2
with an additional at least about 1 amino acid, preferably with an
additional at least about 5 amino acids, preferably with an
additional at least about 10 amino acids, more preferably with an
additional at least about 15 amino acids, more preferably with an
additional at least about 20 amino acids, more preferably with an
additional at least about 30 amino acids, more preferably with an
additional at least about 40 amino acids, even more preferably with
an additional at least about 50 amino acids added to the amino
and/or carboxy termini of SEQ ID NO: 2.
[0042] The invention further provides for a Q3 SPARC deletion
mutant polypeptide wherein conservative substitutions have been
made in the amino acid sequence, including wherein the substituted
amino acids comprise natural and/or non-natural amino acids. In
order to exemplify what is meant by conservative substitution,
Groups A-F are listed below. The replacement of one member of the
following groups by another member of the same group is considered
to be a conservative substitution.
[0043] Group A includes leucine, isoleucine, valine, methionine,
phenylalanine, serine, cysteine, threonine, and modified amino
acids having the following side chains: ethyl, iso-butyl,
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CHOHCH.sub.3 and CH.sub.2SCH.sub.3.
[0044] Group B includes glycine, alanine, valine, serine, cysteine,
threonine, and a modified amino acid having an ethyl side
chain.
[0045] Group C includes phenylalanine, phenylglycine, tyrosine,
tryptophan, cyclohexylmethyl, and modified amino residues having
substituted benzyl or phenyl side chains.
[0046] Group D includes glutamic acid, aspartic acid, a substituted
or unsubstituted aliphatic, aromatic or benzylic ester of glutamic
or aspartic acid (e.g., methyl, ethyl, n-propyl, iso-propyl,
cyclohexyl, benzyl, or substituted benzyl), glutamine, asparagine,
CO--NH-alkylated glutamine or asparagine (e.g., methyl, ethyl,
n-propyl, and iso-propyl), and modified amino acids having the side
chain --(CH.sub.2).sub.3COOH, an ester thereof (substituted or
unsubstituted aliphatic, aromatic, or benzylic ester), an amide
thereof, and a substituted or unsubstituted N-alkylated amide
thereof.
[0047] Group E includes histidine, lysine, arginine,
N-nitroarginine, p-cycloarginine, g-hydroxyarginine,
N-amidinocitruline, 2-amino guanidinobutanoic acid, homologs of
lysine, homologs of arginine, and ornithine.
[0048] Group F includes serine, threonine, cysteine, and modified
amino acids having C1-C5 straight or branched alkyl side chains
substituted with --OH or --SH.
[0049] Groups A-F are exemplary and are not intended to limit the
invention.
[0050] Additionally, the invention provides for isolated Q3 SPARC
deletion mutant polypeptides with one or more amino acid insertions
or deletions of from about 1 to about 5 amino acids, preferably
from about 1 to about 3 amino acids, more preferably 1 amino acid,
in the SEQ ID NO: 1 sequence.
[0051] The invention further provides for an isolated nucleic acid
molecule encoding a Q3 SPARC deletion mutant polypeptide. Suitable
isolated nucleic acids include, without limitation, DNA, RNA, and
peptide-nucleic acids. Isolated nucleic acids encompassed by the
invention include, but are not limited to, nucleic acids comprising
the sequence of SEQ ID NO: 3 without additional 5' or 3'
nucleotides or with an additional at least about 1 nucleotide,
preferably an additional at least about 3 nucleotides, more
preferably at least about 9 nucleotides, more preferably at least
about 20 nucleotides, more preferably at least about 50
nucleotides, more preferably at least about 100 nucleotides, even
more preferably at least about 1,000 nucleotides added to the 5'
and/or 3' ends of SEQ ID NO: 3. The invention also provides for an
isolated nucleic acid molecule encoding a Q3 SPARC deletion mutant
polypeptide wherein the nucleic acid sequence is at least about 80%
homologous to the corresponding sequences of SEQ ID NO: 3,
preferably at least about 90% homologous to the corresponding
sequences of SEQ ID NO: 3, even more preferably at least about 95%
homologous to the corresponding sequences of SEQ ID NO: 3, even
more preferably at least about 99% homologous to the corresponding
sequences of SEQ ID NO: 3. By "corresponding sequences of SEQ ID
NO: 3" it is meant, those sequences which align with the sequences
of SEQ ID NO: 3 wherein the region of alignment is at least about
30 nucleotides long, preferably is at least about 45 nucleotides
long, more preferably is at least about 60 nucleotides long, more
preferably is at least about 90 nucleotides long, more preferably
is at least about 120 nucleotides long, more preferably is at least
about 150 nucleotides long, even more preferably is at least about
300 nucleotides long. Various methods of sequence alignment are
known in the biotechnology arts (see, e.g., Rosenberg, BMC
Bioinformatics 6:278 (2005); Altschul et al., FEBS J. 272(20):
5101-5109 (2005)).
[0052] In addition, the invention provides for an isolated nucleic
acid molecule encoding a Q3 SPARC deletion mutant polypeptide
polypeptide wherein the isolated nucleic acid molecule hybridizes
to SEQ ID NO: 3 under low stringency conditions, preferably under
moderately stringent conditions, even more preferably under highly
stringent conditions. "High stringency conditions" preferably allow
for from about 25% to about 5% mismatch, more preferably from about
15% to about 5% mismatch, and most preferably from about 10% to
about 5% mismatch of the nucleic acid sequence. "Moderately
stringent conditions" preferably allow for from about 40% to about
15% mismatch, more preferably from about 30% to about 15% mismatch,
and most preferably from about 20% to about 15% mismatch of the
nucleic acid sequence. "Low stringency conditions" preferably allow
for from about 60% to about 35% mismatch, more preferably from
about 50% to about 35% mismatch, and most preferably from about 40%
to about 35% mismatch of the nucleic acid sequence.
[0053] The absence of non-specific binding may be tested by the use
of a second target which lacks even a partial degree of
complementarity (e.g., less than about 30% identity); in the
absence of non-specific binding the probe will not hybridize to the
second non-complementary target. There can be partial homology or
complete homology (i.e., identity). A partially complementary
sequence is one that at least partially inhibits a completely
complementary sequence from hybridizing to a target nucleic acid.
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or Northern blot, solution
hybridization and the like) under conditions of low stringency. A
"substantially homologous" sequence or probe will compete for and
inhibit the binding (i.e., the hybridization) of a completely
homologous sequence to a target under conditions of low stringency.
This is not to say that conditions of low stringency are such that
non-specific binding is permitted; low stringency conditions
require that the binding of two sequences to one another be a
specific (i.e., selective) interaction. The term "homology" refers
to a degree of complementarity.
[0054] Exemplary moderate stringency conditions include overnight
incubation at 37.degree. C. in a solution comprising 20% formamide,
5.times.SSC (150 mM NaCl and 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed
by washing in 1.times.SSC at about 37-50.degree. C., or
substantially similar conditions, e.g., the moderately stringent
conditions described in Sambrook & Russell, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York
(2001), pp. 6.1-6.62. High stringency conditions are conditions
that, for example (1) use low ionic strength and high temperature
for washing, such as with a composition comprising 0.015 M sodium
chloride and 0.0015 M sodium citrate, and 0.1% (w/v) sodium dodecyl
sulfate (SDS) at 50.degree. C., (2) employ a denaturing agent
during hybridization, such as a composition comprising formamide,
for example, 50% (v/v) formamide with 0.1% (w/v) bovine serum
albumin (BSA), 0.1% Ficoll, 0.1% polyvinylpyrrolidone (PVP), and 50
mM sodium phosphate buffer at pH 7.5 with 750 mM sodium chloride
and 75 mM sodium citrate at 42.degree. C., or (3) employ a
composition comprising 50% formamide, 5.times.SSC (0.75 M NaCl and
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times.Denhardt's solution, and sonicated
salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate
at 42.degree. C., with washes at (i) 42.degree. C. in
0.2.times.SSC, (ii) at 55.degree. C. in 50% formamide, and (iii) at
55.degree. C. in 0.1.times.SSC (preferably in combination with
EDTA).
[0055] The invention further provides for nucleic acid encoding a
Q3 SPARC deletion mutant polypeptide with conservative amino acid
substitutions including (a) the change of a leucine, isoleucine,
valine, methionine, phenylalanine, serine, cysteine, or threonine
to a leucine, isoleucine, valine, methionine, phenylalanine,
serine, cysteine, or threonine, (b) the change of a glycine,
alanine, valine, serine, cysteine, or threonine to a glycine,
alanine, valine, serine, cysteine, or threonine, (c) the change of
a phenylalanine, tyrosine, or tryptophan to a phenylalanine,
tyrosine, or tryptophan, (d) the change of a glutamic acid,
aspartic acid, glutamine, or asparagine to a glutamic acid,
aspartic acid, glutamine, or asparagine (e) the change of a
histidine, lysine, or arginine to a histidine, lysine, or arginine,
and (f) the change of a serine, threonine, or cysteine to a serine,
threonine, or cysteine, and (g) combinations thereof.
[0056] Additionally, the invention provides for isolated nucleic
acids encoding Q3 SPARC deletion mutant polypeptides with one or
more amino acid insertions or deletions of from about 1 to about 5
amino acids, preferably from about 1 to about 3 amino acids, more
preferably 1 amino acid, in the SEQ ID NO: 1 sequence.
[0057] Mutagenesis can be undertaken by any of several methods
known in the art. Generally, mutagenesis can be accomplished by
cloning the nucleic acid sequence into a plasmid or some other
vector for ease of manipulation of the sequence. Then, a unique
restriction site at which further nucleic acids can be added into
the nucleic acid sequence is identified or inserted into the
nucleic acid sequence. A double-stranded synthetic oligonucleotide
generally is created from overlapping synthetic single-stranded
sense and antisense oligonucleotides such that the double-stranded
oligonucleotide incorporates the restriction sites flanking the
target sequence and, for instance, can be used to incorporate
replacement DNA. The plasmid or other vector is cleaved with the
restriction enzyme, and the oligonucleotide sequence having
compatible cohesive ends is ligated into the plasmid or other
vector to replace the original DNA.
[0058] Other means of in vitro site-directed mutagenesis are known
to those skilled in the art, and can be accomplished (in
particular, using an overlap-extension polymerase chain reaction
(PCR), see, e.g., Parikh & Guengerich, Biotechniques 24:428-431
(1998)). Complementary primers overlapping the site of change can
be used to PCR amplify the whole plasmid in a mixture containing
500 mM dNTPs, 2 units of Pfu polymerase, 250 ng each of sense and
antisense primers, and 200 ng of plasmid DNA comprising a sequence
encoding Q3 SPARC deletion mutant polypeptide. The PCR desirably
involves 18 cycles with an extension time of 2.5 minutes for each
Kb of DNA. The PCR products can be treated with DpnI (which only
digests the adenine-methylated plasmid DNA) and transformed into
Escherichia coli DH5.alpha. cells. Transformants can be screened by
restriction enzyme digestion for incorporation of the changes,
which then can be confirmed by DNA sequence analysis.
[0059] The nucleic acid fragment encoding the mutagenized sequence
can be isolated, e.g., by PCR amplification using 5' and 3'
primers, preferably ones that terminate in further unique
restriction sites, that flank the mutated nucleotide. Use of
primers in this fashion results in an amplified sequence that is
flanked by the unique restriction sites. The unique restriction
sites can be used for further convenient subcloning of the
fragment.
[0060] The invention further provides for a recombinant vector
comprising the nucleic acid sequence encoding a, wherein, e.g., the
vector further comprising a promoter controlling the expression of
the Q3 SPARC deletion mutant polypeptide encoding nucleic acid
sequences. In addition, the invention provides for a cell
comprising the nucleic acid molecule of claim 3, wherein the cell
is a prokaryotic cell or a eukaryotic cell. Methods of tissue
culture are well known to the skilled artisan (see, e.g., Sambrook
& Russell, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, New York (2001), pp. 16.1-16.54).
Accordingly, the invention further provides for method of making
the polypeptide of claim 1 comprising: (a) transforming cells with
a nucleic acid encoding the polypeptide of claim 1; (b) inducing
the expression of the polypeptide by the transformed cells; and (c)
purifying the polypeptide.
[0061] The invention further provides nucleic acid constructs
comprising control elements and a Q3 SPARC deletion mutant
polypeptide nucleic acid molecule described herein operatively
linked to the control elements (e.g., a suitable promoter) for
expression of a Q3 SPARC deletion mutant polypeptide or a
polypeptide herein described with conservative amino acid changes
in a Q3 SPARC deletion mutant polypeptide. Protein expression is
dependent on the level of RNA transcription, which is in turn
regulated by DNA signals. Similarly, translation of mRNA requires,
at the very least, an AUG initiation codon, which is usually
located within 10 to 100 nucleotides of the 5' end of the message.
Sequences flanking the AUG initiator codon have been shown to
influence its recognition by eukaryotic ribosomes, with conformity
to a perfect Kozak consensus sequence resulting in optimal
translation (see, e.g., Kozak, J. Molec. Biol. 196: 947-950
(1987)). Also, successful expression of an exogenous nucleic acid
in a cell can require post-translational modification of a
resultant protein. Accordingly, the invention provides plasmids
encoding Q3 SPARC deletion mutant polypeptides wherein the vector
is, e.g., pcDNA3.1 or a derivative thereof, and including but, not
limited to, the pVT1000Q3 plasmid disclosed herein.
[0062] The nucleic acid molecules described herein preferably
comprise a coding region operatively linked to a suitable promoter,
which promoter is preferably functional in eukaryotic cells. Viral
promoters, such as, without limitation, the RSV promoter and the
adenovirus major late promoter can be used in the invention.
Suitable non-viral promoters include, but are not limited to, the
phosphoglycerokinase (PGK) promoter and the elongation factor
1.alpha. promoter. Non-viral promoters are desirably human
promoters. Additional suitable genetic elements, many of which are
known in the art, also can be ligated to, attached to, or inserted
into the inventive nucleic acid and constructs to provide
additional functions, level of expression, or pattern of
expression. The native promoters for expression of the SPARC family
genes also can be used, in which event they are preferably not used
in the chromosome naturally encoding them unless modified by a
process that substantially changes that chromosome. Such
substantially changed chromosomes can include chromosomes
transfected and altered by a retroviral vector or similar process.
Alternatively, such substantially changed chromosomes can comprise
an artificial chromosome such as a HAC, YAC, or BAC.
[0063] In addition, the nucleic acid molecules described herein may
be operatively linked to enhancers to facilitate transcription.
Enhancers are cis-acting elements of DNA that stimulate the
transcription of adjacent genes. Examples of enhancers which confer
a high level of transcription on linked genes in a number of
different cell types from many species include, without limitation,
the enhancers from SV40 and the RSV-LTR. Such enhancers can be
combined with other enhancers which have cell type-specific
effects, or any enhancer may be used alone.
[0064] To optimize protein production the inventive nucleic acid
molecule can further comprise a polyadenylation site following the
coding region of the nucleic acid molecule. Also, preferably all
the proper transcription signals (and translation signals, where
appropriate) will be correctly arranged such that the exogenous
nucleic acid will be properly expressed in the cells into which it
is introduced. If desired, the exogenous nucleic acid also can
incorporate splice sites (i.e., splice acceptor and splice donor
sites) to facilitate mRNA production while maintaining an inframe,
full length transcript. Moreover, the inventive nucleic acid
molecules can further comprise the appropriate sequences for
processing, secretion, intracellular localization, and the
like.
[0065] The nucleic acid molecules can be inserted into any suitable
vector. Suitable vectors include, without limitation, viral
vectors. Suitable viral vectors include, without limitation,
retroviral vectors, alphaviral, vaccinial, adenoviral,
adenoassociated viral, herpes viral, and fowl pox viral vectors.
The vectors preferably have a native or engineered capacity to
transform eukaryotic cells, e.g., CHO-K1 cells. Additionally, the
vectors useful in the context of the invention can be "naked"
nucleic acid vectors (i.e., vectors having little or no proteins,
sugars, and/or lipids encapsulating them) such as plasmids or
episomes, or the vectors can be complexed with other molecules.
Other molecules that can be suitably combined with the inventive
nucleic acids include without limitation viral coats, cationic
lipids, liposomes, polyamines, gold particles, and targeting
moieties such as ligands, receptors, or antibodies that target
cellular molecules.
[0066] The nucleic acid molecules described herein can be
transformed into any suitable cell, typically a eukaryotic cell,
such as, e.g., CHO, HEK293, or BHK, desirably resulting in the
expression of a Q3 SPARC deletion mutant polypeptide such as, e.g.,
polypeptide comprising of SEQ ID NO: 2 or a variant thereof as
described herein. The cell can be cultured to provide for the
expression of the nucleic acid molecule and, therefore, the
production of the Q3 SPARC deletion mutant polypeptide such as,
e.g., a polypeptide comprising the amino acid sequence of SEQ ID
NO: 2 or a variant thereof as described herein.
[0067] Therefore, the invention provides for a cell transformed or
transfected with an inventive nucleic acid molecule described
herein. Means of transforming, or transfecting, cells with
exogenous DNA molecules are well known in the art. For example,
without limitation, a DNA molecule is introduced into a cell using
standard transformation or transfection techniques well known in
the art such as calcium-phosphate or DEAE-dextran-mediated
transfection, protoblast fusion, electroporation, liposomes and
direct microinjection (see, e.g., Sambrook & Russell, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York (2001), pp. 1.1-1.162, 15.1-15.53, 16.1-16.54). A widely
used method for transformation is transfection mediated by either
calcium phosphate or DEAE-dextran. Depending on the cell type, up
to 20% of a population of cultured cells can be transfected at any
one time.
[0068] Another example of a transformation method is the protoplast
fusion method, protoplasts derived from bacteria carrying high
numbers of copies of a plasmid of interest are mixed directly with
cultured mammalian cells. After fusion of the cell membranes
(usually with polyethylene glycol), the contents of the bacteria
are delivered into the cytoplasm of the mammalian cells, and the
plasmid DNA is transferred to the nucleus. Protoplast fusion is not
as efficient as transfection for many of the cell lines that are
commonly used for transient expression assays, but it is useful for
cell lines in which endocytosis of DNA occurs inefficiently.
Protoplast fusion frequently yields multiple copies of the plasmid
DNA randomly integrated into the host chromosome.
[0069] Electroporation, the application of brief, high-voltage
electric pulses to a variety of mammalian and plant cells leads to
the formation of nanometer-sized pores in the plasma membrane. DNA
is taken directly into the cell cytoplasm either through these
pores or as a consequence of the redistribution of membrane
components that accompanies closure of the pores. Electroporation
can be extremely efficient and can be used both for transient
expression of clones genes and for establishment of cell lines that
carry integrated copies of the gene of interest.
[0070] Liposome transformation involves encapsulation of DNA and
RNA within liposomes, followed by fusion of the liposomes with the
cell membrane. In addition, DNA that is coated with a synthetic
cationic lipid can be introduced into cells by fusion.
Alternatively, linear and/or branched polyethylenimine (PEI) can be
used in transfection.
[0071] Direct microinjection of a DNA molecule into nuclei has the
advantage of not exposing the DNA molecule to cellular compartments
such as low-pH endosomes. Microinjection is, therefore, used
primarily as a method to establish lines of cells that carry
integrated copies of the DNA of interest.
[0072] Such techniques can be used for both stable and transient
transformation of eukaryotic cells. The isolation of stably
transformed cells requires the introduction of a selectable marker
in conjunction with the transformation with the gene of interest.
Such selectable markers include genes which confer resistance to
neomycin as well as the HPRT gene in HPRT negative cells. Selection
can require prolonged culture in selection media, at least for
about 2-7 days, preferable for at least about 1-5 weeks (see, e.g.,
Sambrook & Russell, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York (2001), pp.
16.1-16.54).
[0073] Nucleic acid sequences for use in the present invention may
also be produced in part or in total by chemical synthesis, e.g. by
the phosphoramidite method described by Beaucage, et al. (Tetra.
Letts. 22: 1859-1862 (1987)), or the triester method (Matteucci et
al., J. Am. Chem. Soc. 103: 3185 (1981)), which may be performed on
commercial automated oligonucleotide synthesizers. A
double-stranded fragment may be obtained from the single-stranded
product of chemical synthesis either by synthesizing the
complementary strand and annealing the strand together under
appropriate conditions, or by synthesizing the complementary strand
using DNA polymerase with an appropriate primer sequence.
[0074] Gene Therapy Gene therapy is a medical intervention that
involves modifying the genetic material of living cells to fight
disease. Gene therapy is being studied in clinical trials (research
studies with humans) for many different types of cancer and for
other diseases.
[0075] The invention further provides for an isolated nucleic acid
molecule encoding a Q3 SPARC deletion mutant polypeptide suitable
for use in "gene therapy" (see, e.g., Patil et al., AAPS J.
7(1):E61-77 (2005)). Suitable nucleic acid include, but are not
limited to nucleic acids comprising SEQ ID NO: 3 or modifications
and homologues thereof as described herein. One of the goals of
gene therapy is to supply cells with altered genes, such as, e.g.,
an isolated nucleic acid molecule encoding a Q3 SPARC deletion
mutant polypeptide. Gene therapy is also being studied as a way to
change how a cell functions; for example, by stimulating immune
system cells to attack cancer cells.
[0076] In general, a gene is delivered to the cell using a "vector"
such as those disclosed herein. The most common types of vectors
used in gene therapy are viruses. Viruses used as vectors in gene
therapy are genetically disabled; they are unable to reproduce
themselves. Most gene therapy clinical trials rely on mouse
retroviruses to deliver the desired gene. Other viruses used as
vectors include adenoviruses, adeno-associated viruses, poxviruses,
and the herpes virus. Suitable viral gene therapy vectors and modes
of their administration in vivo and ex vivo are know in the
art.
[0077] Gene therapy can be done both ex vivo and in vivo.
Typically, in ex vivo gene therapy clinical trials, cells from the
patient's blood or bone marrow are removed and grown in the
laboratory. The cells are exposed to the virus that is carrying the
desired gene. The virus enters the cells, and the desired gene
becomes part of the cells' DNA. The cells grow in the laboratory
and are then returned to the patient by injection into a vein. In
in vivo gene therapy, vectors such as, e.g., viruses or liposomes
are used to deliver the desired gene to cells inside the patient's
body.
[0078] A Q3 SPARC deletion mutant polypeptide can be expressed and
purified from a recombinant host cell. Recombinant host cells may
be prokaryotic or eukaryotic, including but not limited to bacteria
such as E. coli, fungal cells such as yeast, insect cells including
but, not limited to, drosophila and silkworm derived cell lines,
and mammalian cells and cell lines. When expressing a Q3 SPARC
deletion mutant polypeptide in a cell, e.g., a human cell, whether,
in vitro or in vivo, the codons selected for such the
polynucleotide encoding the Q3 SPARC can be optimized for a given
cell type (i.e., species). Many techniques for codon optimization
are known in the art (see, e.g., Jayaraj et al, Nucleic Acids Res.
33(9):3011-6 (2005); Fuglsang et al., Protein Expr. Purif.
31(2):247-9 (2003); Wu et al., "The Synthetic Gene Designer: a
Flexible Web Platform to Explore Sequence Space of Synthetic Genes
for Heterologous Expression," csbw, 2005 IEEE Computational Systems
Bioinformatics Conference--Workshops (CSBW '05), pp. 258-259
(2005)).
[0079] In certain embodiments, when expressing and purifying a Q3
SPARC deletion mutant polypeptide, techniques for improving protein
solubility are employed to prevent the formation of inclusion body
(which are insoluble fractions), and therefore obtaining large
quantities of the polypeptide. SPARC accumulated in inclusion
bodies is an inactive-type SPARC not retaining its physiological
activities.
[0080] Solubility of a purified Q3 SPARC deletion mutant
polypeptide can be improved by methods known in the art. For
example, solubility may also be improved by expressing a functional
fragment, but not the full length Q3 SPARC deletion mutant
polypeptide. In addition, to increase the solubility of an
expressed protein (e.g., in E. coli), one can reduce the rate of
protein synthesis by lowering the growth temperature, using a
weaker promoter, using a lower copy number plasmid, lowering the
inducer concentration, changing the growth medium as described in
Georgiou & Valax (Current Opinion Biotechnol. 7:190-197
(1996)). This decreases the rate of protein synthesis and usually
more soluble protein is obtained. One can also add prostethic
groups or co-factors which are essential for proper folding or for
protein stability, or add buffer to control pH fluctuation in the
medium during growth, or add 1% glucose to repress induction of the
lac promoter by lactose, which is present in most rich media (such
as LB, 2.times.YT). Polyols (e.g., sorbitol) and sucrose may also
be added to the media because the increase in osmotic pressure
caused by these additions leads to the accumulation of
osmoprotectants in the cell, which stabilize the native protein
structure. Ethanol, low molecular weight thiols and disulfides, and
NaCl may be added. In addition, chaperones and/or foldases may be
co-expressed with the desired polypeptide. Molecular chaperones
promote the proper isomerization and cellular targeting by
transiently interacting with folding intermediates. E. coli
chaperone systems include but, are not limited to: GroES-GroEL,
DnaK-DnaJ-GrpE, CIpB.
[0081] Foldases accelerate rate-limiting steps along the folding
pathway. Three types of foldases play an important role: peptidyl
prolyl cis/trans isomerases (PPI's), disulfide oxidoreductase
(DsbA) and disulfide isomerase (DsbC), protein disulfide isomerase
(PDI) which is an eukaryotic protein that catalyzes both protein
cysteine oxidation and disulfide bond isomerization. Co-expression
of one or more of these proteins with the target protein could lead
to higher levels of soluble target protein.
[0082] A Q3 SPARC deletion mutant polypeptide can be produced as a
fusion protein in order to improve its solubility and production.
The fusion protein comprises a Q3 SPARC deletion mutant polypeptide
and a second polypeptide fused together in frame. The second
polypeptide may be a fusion partner known in the art to improve the
solubility of the polypeptide to which it is fused, for example,
NusA, bacterioferritin (BFR), GrpE, thioredoxin (TRX) and
glutathione-S-- transferase (GST). Novagen Inc. (Madison, Wis.)
provides the pET 43.1 vector series which permit the formation of a
NusA-target fusion. DsbA and DsbC have also shown positive effects
on expression levels when used as a fusion partner, therefore can
be used to fuse with a SPARC polypeptide for achieving higher
solubility.
[0083] In one embodiment, a Q3 SPARC deletion mutant polypeptide is
produced as a fusion polypeptide comprising the Q3 SPARC deletion
mutant polypeptide and a fusion partner thioredoxin, as described
in U.S. Pat. No. 6,387,664, hereby incorporated by reference in its
entirety. The thioredoxin-SPARC fusion can be produced in E. coli
as an easy-to-formulate, soluble protein in a large quantity
without losing the physiological activities. Although U.S. Pat. No.
6,387,664 provides a fusion SPARC protein with SPARC fused to the
C-terminus of thioredoxin, it is understood, for the purpose of the
present invention, a Q3 SPARC deletion mutant polypeptide can be
fused either to the N-terminus or the C-terminus of a second
polypeptide, so long as its sensitizing function is retained.
[0084] In addition to increase solubility, a fusion protein
comprising a Q3 SPARC deletion mutant polypeptide can be
constructed for the easy detection of the expression of the Q3
SPARC deletion mutant polypeptidein a cell. In one embodiment, the
second polypeptide which fused to the Q3 SPARC deletion mutant
polypeptideis a reporter polypeptide. The reporter polypeptide,
when served for such detection purpose, does not have to be fused
with the Q3 SPARC deletion mutant polypeptide. It may be encoded by
the same polynucleotide (e.g., a vector) which also encodes the Q3
SPARC deletion mutant polypeptide and be co-introduced and
co-expressed in a target cell.
[0085] Preferably, the reporter polypeptide used in the invention
is an autofluorescent protein (e.g., GFP, EGFP). Autofluorescent
proteins provide a ready assay for identification of expression of
a polynucleotide (and the polypeptide product) of interest. Because
the activity of the reporter polypeptide (and by inference its
expression level) can be monitored quantitatively using a flow
sorter, it is simple to assay many independent transfectants either
sequentially or in bulk population. Cells with the best expression
can then be screened for or selected from the population. This is
useful when selecting a recombinant cell comprising a Q3 SPARC
deletion mutant polypeptideor polynucleotide for sensitizing
treatment according to the present invention.
[0086] Quantitative parameters such as mean fluorescence intensity
and variance can be determined from the fluorescence intensity
profile of the cell population (Shapiro, H., 1995, Practical Flow
Cytometry, 217-228). Non-limiting examples of reporter molecules
useful in the invention include luciferase (from firefly or other
species), chloramphenicol acetyltransferase, .beta.-galactosidase,
green fluorescent protein (GFP), enhanced green fluorescent protein
(EGFP), and dsRed.
[0087] Expression of the SPARC polypeptide (either by itself, or as
a fusion protein) can also be directly determined by an immunoassay
such as an ELISA (enzyme-linked immunoabsorbent assay) (see, e.g.,
U.S. Pat. Nos. 5,962,320; 6,187,307; and 6,194,205), Western blot,
or by other methods routine in the art. The expression of a Q3
SPARC deletion mutant polypeptide can be indirectly detected by
detecting the transcript of the protein (e.g., by hybridization
analysis such as Northern blot or DNA Microarray, or by PCR).
[0088] In one embodiment, a polynucleotide encoding a second
polypeptide is fused to a polynucleotide encoding a Q3 SPARC
deletion mutant polypeptide through an intervening linker sequence
which encodes for a linker polypeptide.
[0089] In another embodiment, the linker polypeptide comprises a
protease cleavage site comprising a peptide bond which is
hydrolyzable by a protease. As a result, the Q3 SPARC deletion
mutant polypeptide can be separated from the second polypeptide
after expression by proteolysis. The linker can comprise one or
more additional amino acids on either side of the bond to which the
catalytic site of the protease also binds (see, e.g., Schecter
& Berger, Biochem. Biophys. Res. Commun. 27, 157-62 (1967)).
Alternatively, the cleavage site of the linker can be separate from
the recognition site of the protease and the two cleavage site and
recognition site can be separated by one or more (e.g., two to
four) amino acids. In one aspect, the linker comprises at least
about 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 20, about 30, about
40, about 50 or more amino acids. More preferably the linker is
from about 5 to about 25 amino acids in length, and most
preferably, the linker is from about 8 to about 15 amino acids in
length.
[0090] Some proteases useful according to the invention are
discussed in the following references: Hooper et al., Biochem. J.
321: 265-279 (1997); Werb, Cell 91: 439-442 (1997); Wolfsberg et
al., J. Cell Biol. 131: 275-278 (1995); Murakami & Etlinger,
Biochem. Biophys. Res. Comm. 146: 1249-1259 (1987); Berg et al.,
Biochem. J. 307: 313-326 (1995); Smyth and Trapani, Immunology
Today 16: 202-206 (1995); Talanian et al., J. Biol. Chem. 272:
9677-9682 (1997); and Thornberry et al., J. Biol. Chem. 272:
17907-17911 (1997). Cell surface proteases also can be used with
cleavable linkers according to the invention and include, but are
not limited to: Aminopeptidase N; Puromycin sensitive
aminopeptidase; Angiotensin converting enzyme; Pyroglutamyl
peptidase II; Dipeptidyl peptidase IV; N-arginine dibasic
convertase; Endopeptidase 24.15; Endopeptidase 24.16; Amyloid
precursor protein secretases alpha, beta and gamma; Angiotensin
converting enzyme secretase; TGF alpha secretase; TNF alpha
secretase; FAS ligand secretase; TNF receptor-1 and -II secretases;
CD30 secretase; KL1 and KL2 secretases; IL6 receptor secretase;
CD43, CD44 secretase; CD16-I and CD16-II secretases; L-selectin
secretase; Folate receptor secretase; MMP 1, 2, 3, 7, 8, 9, 10, 11,
12, 13, 14, and 15; Urokinase plasminogen activator; Tissue
plasminogen activator; Plasmin; Thrombin; BMP-1 (procollagen
C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and,
Granzymes A, B, C, D, E, F, G, and H.
[0091] An alternative to relying on cell-associated proteases is to
use a self-cleaving linker. For example, the foot and mouth disease
virus (FMDV) 2A protease may be used as a linker. This is a short
polypeptide of 17 amino acids that cleaves the polyprotein of FMDV
at the 2A/2B junction. The sequence of the FMDV 2A propeptide is
NFDLLKLAGDVESNPGP. Cleavage occurs at the C-terminus of the peptide
at the final glycine-proline amino acid pair and is independent of
the presence of other FMDV sequences and cleaves even in the
presence of heterologous sequences.
[0092] Insertion of this sequence between two protein coding
regions (i.e., between the Q3 SPARC deletion mutant polypeptide and
the second polypeptide of a fusion protein according to the
invention) results in the formation of a self-cleaving chimera
which cleaves itself into a C-terminal fragment which carries the
C-terminal proline of the 2A protease on its N-terminal end, and an
N-terminal fragment that carries the rest of the 2A protease
peptide on its C-terminus (see, e.g., de Felipe et al., Gene
Therapy 6: 198-208 (1999)). Self-cleaving linkers and additional
protease-linker combinations are described further in PCT
Publication WO 01/20989, the entirety of which is incorporated by
reference herein.
[0093] Polynucleotides encoding linker sequences described above
can be cloned from sequences encoding the natural substrates of an
appropriate protease or can be chemically synthesized using methods
routine in the art.
[0094] Affinity chromatography employing SPARC ligands and/or
anti-SPARC antibodies can also by used to purify SPARC polypeptides
in accordance with the invention. Affinity chromatography can be
used alone or in conjunction with ion-exchange, molecular sizing,
or HPLC chromatographic techniques. Such chromatographic approach
can be performed using columns or in batch formats. Such
chromatographic purification methods are well known in the art.
[0095] The invention provides for the use of antibody raised
against SPARC, in particular a Q3 SPARC deletion mutant
polypeptide, as therapeutic agent against, or imaging agent for,
diseases where SPARC plays a role. Antibodies can be tested for
specificity of binding by comparing binding to appropriate antigen
to binding to irrelevant antigen or antigen mixture under a given
set of conditions. If the antibody binds to the appropriate antigen
at least about 2 times, preferably at least about 5 times, more
preferably at least about 7 times, and more preferably at least
about 10 times more strongly than to irrelevant antigen, including,
e.g., a wild type SPARC polypeptide, or antigen mixture then it is
considered to be specific. In addition, the invention provides for
an antibody against a SPARC polypeptide, such as, e.g., a Q3 SPARC
deletion mutant polypeptide, wherein the antibody binds both Q3
SPARC deletion mutant polypeptides and wild type SPARC polypeptides
but, wherein the antibody binds to a Q3 SPARC deletion mutant
polypeptide at least about 2 times, preferably at least about 5
times, more preferably at least about 7 times, and more preferably
at least about 10 times more strongly than to wild type SPARC
polypeptides. Various methods of determining antibody binding
strength are known to those of ordinary skill in the art (see,
e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold
Spring Harbor (1988)).
[0096] Any suitable antibodies against a SPARC polypeptide, such
as, e.g., a Q3 SPARC deletion mutant polypeptide, can be used in
the inventive method, so long as the antibody exhibits specific
binding to a SPARC polypeptide, such as, e.g., Q3 SPARC deletion
mutant polypeptide. The antibody can either be monoclonal or
polyclonal; and can be produced either through immunization of an
animal or produced through recombinant DNA technology such as phage
display and in vitro mutagenesis or synthesis of the variable
regions of the antibody heavy and light chain genes. Polyclonal
antibodies include, but are not limited to, human antibodies and
humanized antibodies derived from animals such as avian (e.g,
chicken), rodent (e.g., rat, mouse, hamster, guinea pig), cow,
goat, sheep, rabbit and the like. Monoclonal antibody include
antibody derived from a single clone of antibody producing cells
including, but not limited to, human cells, and antibodies derived
from the cells of other animal types, such as, for example,
chicken, rabbit, rat, mouse, hamster, guinea pig, cow, goat, sheep,
and the like. Synthetic antibody includes antibody produced using
recombinant DNA means via genetic engineering of the variable
regions of the heavy and light chain genes. Synthetic antibody also
includes chemically synthesized antibody fragment with Q3 SPARC
deletion mutant polypeptide polypeptide binding activity or
antibody derived from phage display or similar technology. Methods
of making antibodies are well known in the art (see, e.g., Harlow
& Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor,
pp. 1-420 (1988)).
[0097] For human use, in order to avoid immunogenicity and an
immune response, it is preferable to use humanized anti-Q3 SPARC
deletion mutant polypeptide polypeptide antibody or suitable
fragments such as Fc or Fab. Humanized antibody or fragments
thereof are produced, for example, using one of the following
established methods: (1) Humanized antibody can be constructed
using human IgG backbone replacing the variable CDR region with
that of antibody against SPARC, the heavy and light chain are
independently expressed under separate promoters or coexpressed
under one promoter with IRES sequence; (2) Humanized monoclonal
antibody is raised against SPARC using mouse engineered to have
human immune system; or (3) Humanized antibody against SPARC is
raised using phagemid (M13, lambda coliphage, or any phage system
capable of surface presentation). To construct the full length
antibody, the variable region is transferred onto the CDR of both
Heavy chain and Light chain. The coexpression of the Heavy chain
and Light Chain in mammalian cells such as CHO, 293, or human
myeloid cells results in full length antibody. Similarly, Fc or Fab
fragments and single chain antibodies can be prepared using well
established methods.
[0098] The antibody against a SPARC polypeptide, e.g., a Q3 SPARC
deletion mutant polypeptide made in accordance with the invention,
is also not limited to a whole antibody or fragment of the antibody
retaining the binding site for the SPARC polypeptide, e.g., for a
Q3 SPARC deletion mutant polypeptide (e.g., Fab and Fab2). The
antibody is also not limited to any one class of antibody, e.g.,
IgM, IgA, IgG, IgE, IgD, and IgY. The antibody is also not limited
to divalent antibody, monovalent, or chimeria with one valent for
SPARC and another for an effector such tTF or ricin A. The
humanized antibody is not limited to IgG. The same technologies can
be used to generate all other classes of antibodies such as IgE,
IgA, IgD, IgM, each has different ADCC and CDC activities
appropriate to particular disease target. Functional fragment of
the antibody can be generated by limited proteolysis. These
fragments can be monovalent such as Fab' or divalent, such as Fab2.
Fragments can also be synthesized as single chain scfv or diabodies
in E. coli.
[0099] The invention also provides a method for destruction of
SPARC expression tissues such as tumor and restenotic tissues via
the complement fixation and/or recruitment of cell mediated immune
response by anti-Q3 SPARC deletion mutant polypeptide antibody. In
this case, like that of Rituxan--an anti-CD20 antibody, the
effector moiety is the Fc fragment which can mediate either
complement activation with direct destruction of SPARC expression
cells or recruitment of immune cells to the SPARC expression tissue
with resulting tissue destruction via cell mediate immune
response.
[0100] The invention also provides a method for inhibition of SPARC
activity using neutralizing antibody against SPARC, e.g. an anti-Q3
SPARC deletion mutant polypeptide polypeptide antibody.
Neutralizing antibody has the ability to block the interaction of
SPARC with its effectors in vivo, for example, the interaction of
SPARC with cell surface component or the binding of SPARC to its
natural ligands such as albumin, growth factors, and Ca.sup.2+.
[0101] The invention provides a method for determining the response
of a human or other mammalian tumor to anti-SPARC therapy (e.g.,
anti-Q3 SPARC deletion mutant polypeptide therapy). The method
comprises (a) isolating a biological sample from the human, (b)
detecting the expression of SPARC protein (including, e.g., a Q3
SPARC deletion mutant polypeptide) in the biological sample, and
(c) quantifying the amount of SPARC protein in the biological
sample. As anti-SPARC therapy relies on the binding of SPARC
antibody to SPARC in disease tissue, the presence of SPARC in
disease tissue is necessary for activity.
[0102] Any suitable chemotherapeutic agent can be used to couple
(or conjugate) to a Q3 SPARC deletion mutant polypeptide or an
antibody against a SPARC polypeptide, such as, e.g., a Q3 SPARC
deletion mutant polypeptide, in the inventive method. Suitable
chemotherapeutics include, but are not limited to, tyrosine kinase
inhibitors (genistein), biologically active agents (TNF, of tTF),
radionuclides (.sup.131I, .sup.90Y, .sup.111In, .sup.211At,
.sup.32P and other known therapeutic radionuclides), adriamycin,
ansamycin antibiotics, asparaginase, bleomycin, busulphan,
cisplatin, carboplatin, carmustine, capecitabine, chlorambucil,
cytarabine, cyclophosphamide, camptothecin, dacarbazine,
dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin,
etoposide, epothilones, floxuridine, fludarabine, fluorouracil,
gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan,
lomustine, mechlorethamine, mercaptopurine, meplhalan,
methotrexate, rapamycin (sirolimus) and derivatives, mitomycin,
mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate,
pentostatin, plicamycin, procarbazine, rituximab, streptozocin,
teniposide, thioguanine, thiotepa, taxanes, vinblastine,
vincristine, vinorelbine, taxol, combretastatins, discodennolides,
transplatinum, anti-vascular endothelial growth factor compounds
("anti-VEGFs"), anti-epidermal growth factor receptor compounds
("anti-EGFRs"), 5-fluorouracil, and the like. A dose of one or more
chemotherapeutic agents can be administered according to the
inventive method. The type and number of chemotherapeutic agents
used in the inventive method will depend on the standard
chemotherapeutic regimen for a particular tumor type. In other
words, while a particular cancer may be treated routinely with a
single chemotherapeutic agent, another may be treated routinely
with a combination of chemotherapeutic agents.
[0103] The terms "treating," "treatment," "therapy," and
"therapeutic treatment" as used herein refer to curative therapy,
prophylactic therapy, or preventative therapy. An example of
"preventative therapy" is the prevention or lessening the chance of
a targeted disease (e.g., cancer or other proliferative disease) or
related condition thereto. Those in need of treatment include those
already with the disease or condition as well as those prone to
have the disease or condition to be prevented. The terms
"treating," "treatment," "therapy," and "therapeutic treatment" as
used herein also describe the management and care of a mammal for
the purpose of combating a disease, or related condition, and
includes the administration of a composition to alleviate the
symptoms, side effects, or other complications of the disease,
condition. Therapeutic treatment for cancer includes, but is not
limited to, surgery, chemotherapy, radiation therapy, gene therapy,
and immunotherapy.
[0104] By "therapeutically effective amount" it is meant an amount
that relieves (to some extent, as judged by a skilled medical
practitioner) one or more symptoms of the disease or condition in a
mammal. Additionally, by "therapeutically effective amount" is
meant an amount that returns to normal, either partially or
completely, physiological or biochemical parameters associated with
or causative of a disease or condition. A clinician skilled in the
art can determine the therapeutically effective amount of a
composition in order to treat or prevent a particular disease
condition, or disorder when it is administered, such as
intravenously, subcutaneously, intraperitoneally, orally, or
through inhalation. The precise amount of the composition required
to be therapeutically effective will depend upon numerous factors,
e.g., such as the specific activity of the active agent, the
delivery device employed, physical characteristics of the agent,
purpose for the administration, in addition to many patient
specific considerations. The determination of amount of a
composition that must be administered to be therapeutically
effective is routine in the art and within the skill of an
ordinarily skilled clinician.
[0105] As used herein, the term "agent" or "drug" or "therapeutic
agent" refers to a chemical compound, a mixture of chemical
compounds, a biological macromolecule, or an extract made from
biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues that are suspected of
having therapeutic properties. The agent or drug may be purified,
substantially purified or partially purified. An "agent", according
to the present invention, also includes a radiation therapy
agent.
[0106] As used herein, the term "toxin" refers to a poisonous
substance that is a specific product of the metabolic activities of
a living organism including bacterial, plant, and other toxins,
such as, e.g., diphtheria toxin, pseudomonas exotoxin A,
staphylococcal enterotoxin A, abrin-A toxin, ricin A
(deglycosylated ricin A and native ricin A), TGF-alpha toxin,
cytotoxin from chinese cobra (naja naja atra), and gelonin (a plant
toxin); ribosome inactivating proteins from plants, bacteria and
fungi, such as restrictocin (a ribosome inactivating protein
produced by Aspergillus restrictus) or saporin (a ribosome
inactivating protein from Saponaria officinalis).
[0107] As used herein, the term "proliferative disease" refers to
disease characterized by cellular proliferation (e.g., mitosis)
including both clonal proliferations (e.g., cancer) and polyclonal
proliferations (e.g., hyperplasias; including but, not limited, to
endometriosis, endometrial hyperplasia, benign prostatic
hyperplasia (BPH)).
[0108] As used herein biological molecules are molecules produced
by the metabolic activities of a living organism that have
biological activities such as, e.g., cytokines interleukins, TNF,
or tissue factor (TF). As used herein biologic molecules include
other molecules composed of subunits normally found in living cells
or their analogs such RNA that can have biologic activities such
as, e.g., anti-sense RNA, RNAi, ribozymes, inhibitory
peptide-nucleic acids, and the like.
[0109] Methods for coupling or conjugation of suitable
therapeutics, chemotherapeutics, radionuclides, polypeptides, and
the like to antibodies or fragments thereof are well described in
the art. For example, The invention provides for Q3 SPARC deletion
mutant polypeptide or anti-Q3 SPARC deletion mutant polypeptide
antibodies conjugates, such as, e.g., SPARC-radioinuclide,
SPARC-drug, SPARC-immunomodulator or SPARC-toxin conjugates. Any
suitable method can be used in accordance with the invention to
form the SPARC conjugates. For example, without limitation, free
amino groups in SPARC proteins, such the epsilon-amino group of
lysine, can be conjugated with reagents such as carodiimides or
heterobiofunctional agents. Alternatively, e.g., SPARC suflhydryl
groups can be used for conjugation. In addition, sugar moieties
bound to SPARC glycoproteins or an anti-SPARC antibodies, e.g., an
anti-Q3 SPARC deletion mutant polypeptide antibodies can be
oxidized to form aldehydes groups useful in a number of coupling
procedures known in the art. The conjugates formed in accordance
with the invention can be stable in vivo or labile, such as
enzymatically degradeable tetrapeptide linakages or acid-labile
cis-aconityl or hydrazone linkages.
[0110] The invention also provides for SPARC-carrier-therapeutic
agent molecule or anti-SPARC polypeptide, such as, e.g., anti-Q3
SPARC deletion mutant polypeptide antibody, conjugates such as,
e.g., conjugates of a SPARC and a drug with carriers. Suitable
carriers for use in accordance with the invention include, without
limitation, dextran, human serum albumin, and
hydroxypropylmethacrylamide. Further, the invention provides for
SPARC or anti-SPARC antibody, e.g., an anti-Q3 SPARC deletion
mutant polypeptide antibody, coated drug carrier structures such
as, e.g., liposomes.
[0111] The invention provides for SPARC molecules, including SPARC
polypeptides and proteins and anti-SPARC antibodies, such as, e.g.,
ant-Q3 SPARC deletion mutant polypeptide antibodies, conjugated to
polyethylene glycol (PEG). PEG conjugation can increase the
circulating half-life of a protein, reduce the protein's
immunogenicity and antigenicity, and improve the bioactivity. Any
suitable method of conjugation can be used, including but not
limited to, e.g., reacting methoxy-PEG with a SPARC protein's
available amino groups or other reactive sites such as, e.g.,
histidines or cysteines. In addition, recombinant DNA approaches
may be used to add amino acids with PEG-reactive groups to the
inventive SPARC molecules and antibodies. PEG can be processed
prior to reacting it with the inventive SPARC protein, e.g., linker
groups may be added to the PEG. Further, releasible and hybrid
PEG-ylation strategies may be used in accordance with the
invention, such as, e.g., the PEG-ylation of SPARC such that the
PEG molecules added to certain sites in the SPARC molecule are
released in vivo. Such PEG conjugation methods are known in the art
(See, e.g., Greenwald et al., Adv. Drug Delivery Rev. 55:217-250
(2003)).
[0112] In addition, the invention provides for SPARC fusion
proteins, including, for example without limitation, SPARC
sequences are fused upstream or downstream of diagnostically useful
protein domains (such as hapten, GFP), immunologically active
protein domains (e.g., TF or TNF) or toxin domains.
[0113] The invention also provides for method of the use of
diagnostic agents coupled or conjugated to the SPARC recognition
groups, such as the antibodies, including anti-Q3 SPARC deletion
mutant polypeptide antibodies, or fragments thereof, as described
above. The diagnostic agents include radioisotopes or
radionuclides, MRI contrast agents, X-ray contrast agents,
ultrasound contrast agents and PET contrast agents. Methods
utilized for conjugation are known in the art.
[0114] The expression of SPARC protein in the sample can be
detected and quantified by any suitable method known in the art.
Suitable methods of protein detection and quantification include
Western blot, enzyme-linked immunosorbent assay (ELISA), silver
staining, the BCA assay (Smith et al., Anal. Biochem. 150, 76-85
(1985)), the Lowry protein assay (described in, e.g., Lowry et al.,
J. Biol. Chem. 193, 265-275 (1951)), which is a colorimetric assay
based on protein-copper complexes, and the Bradford protein assay
(described in, e.g., Bradford et al., Anal. Biochem. 72, 248
(1976)), which depends upon the change in absorbance in Coomassie
Blue G-250 upon protein binding. Tumor biopsy can be analyzed by
any of the preceding methods or it can be analyzed by
immunohistochemistry using an anti-SPARC antibody, e.g., an anti-Q3
SPARC deletion mutant polypeptide antibody, (either monoclonal or
polyclonal) in conjunction with appropriate visualization system
(i.e., HRP substrate and HRP-conjugated secondary antibody).
[0115] The types of tumor to be detected, sensitized, and/or
treated in accordance with the invention are generally those found
in humans and other mammals. The tumors can be the result of
inoculation as well, such as in laboratory animals. Many types and
forms of tumors are encountered in the human and other animal
condition, and there is no intention to limit the application of
the methods of the present to any particular tumor type or variety.
Tumors, as is known, include an abnormal mass of tissue that
results from uncontrolled and progressive cell division, and is
also typically known as a "neoplasm." The inventive methods are
useful for tumor cells and associated stromal cells, solid tumors
and tumors associated with soft tissue, such as, soft tissue
sarcoma, for example, in a human. The tumor or cancer can be
located in the oral cavity and pharynx, the digestive system, the
respiratory system, bones and joints (e.g., bony metastases), soft
tissue, the skin (e.g., melanoma), breast, the genital system, the
urinary system, the eye and orbit, the brain and central nervous
system (e.g., glioma), or the endocrine system (e.g., thyroid) and
is not necessarily the primary tumor or cancer. Tissues associated
with the oral cavity include, but are not limited to, the tongue
and tissues of the mouth. Cancer can arise in tissues of the
digestive system including, for example, the esophagus, stomach,
small intestine, colon, rectum, anus, liver, gall bladder, and
pancreas. Cancers of the respiratory system can affect the larynx,
lung, and bronchus and include, for example, non-small cell lung
carcinoma. Tumors can arise in the uterine cervix, uterine corpus,
ovary vulva, vagina, prostate, testis, and penis, which make up the
male and female genital systems, and the urinary bladder, kidney,
renal pelvis, and ureter, which comprise the urinary system. The
tumor or cancer can be located in the head and/or neck (e.g.,
laryngeal cancer and parathyroid cancer). The tumor or cancer also
can be located in the hematopoietic system or lymphoid system, and
include, for example, lymphoma (e.g., Hodgkin's disease and
Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute
lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid
leukemia, chronic myeloid leukemia, and the like). Preferably, the
tumor is located in the bladder, liver, ovary, kidney, gut, brain,
or breast.
[0116] The invention further provides for a method of sensitizing a
disease to a treatment comprising administering a Q3 SPARC deletion
mutant polypeptide and a pharmaceutically acceptable carrier to a
patient. Suitable such diseases in need of sensitization, include,
without limitation, therapy-resistant (e.g., chemotherapy or
radiation therapy-resistant) cancers. As used herein, the term
"sensitizing" refers to an increased sensitivity or reduce the
resistance of a cancer sample or a mammal responding to a
therapeutic treatment. An increased sensitivity or a reduced
sensitivity to a therapeutic treatment is measured according to a
known method in the art for the particular treatment and methods
described herein below, including, but not limited to, cell
proliferative assays or cell death assays. The sensitivity or
resistance may also be measured in animal by measuring the tumor
size reduction over a period of time, for example, 6 month for
human and 4-6 weeks for mouse. A composition or a method sensitizes
response to a therapeutic treatment if the increase in treatment
sensitivity or the reduction in resistance is about 25% or more,
for example, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, or more, to about 2-fold, about 3-fold, about 4-fold,
about 5-fold, about 10-fold, about 15-fold, about 20-fold or more,
compared to treatment sensitivity or resistance in the absence of
such composition or method. The determination of sensitivity or
resistance to a therapeutic treatment is routine in the art and
within the skill of an ordinarily skilled clinician. As such, using
SPARC for disease sensitization according the invention can further
comprise administering one or more non-SPARC therapeutic agents,
e.g., combination chemotherapy or other drugs.
[0117] The invention also provides a method for delivering a
chemotherapeutic agent to a tumor in a mammal. The method comprises
administering to a human or other mammal a therapeutically
effective amount of a delivery agent, such as a pharmaceutical
composition, wherein the delivery agent (e.g., pharmaceutical
composition) comprises the chemotherapeutic agent coupled to the Q3
SPARC deletion mutant polypeptide or anti-Q3 SPARC deletion mutant
polypeptide antibody. For example, the chemotherapeutic agent can
be coupled to a SPARC recognition group such as an antibody
recognizing SPARC protein, or the SPARC antibody alone.
Pharmaceutical compositions preferably include the chemotherapeutic
agent coupled to the SPARC recognition group and a pharmaceutically
acceptable carrier. Descriptions of the chemotherapeutic agent,
tumor, mammal, and components thereof, set forth above in
connection with other embodiments of the invention also are
applicable to those same aspects of the aforesaid method of
delivering a chemotherapeutic agent to a tumor.
[0118] In another preferred embodiment, the invention also provides
a method for delivering a pharmaceutically active agent (such as,
e.g., anti-SPARC or anti-Q3 SPARC deletion mutant polypeptide
antibody alone or chemotherapeutic agent coupled or conjugated to
anti-SPARC or anti-Q3 SPARC deletion mutant polypeptide antibody
and the like) to a site of disease that is characterized by
overexpression of SPARC in a human, or other animal that expresses
such protein or marker. Such diseases include abnormal conditions
of proliferation, tissue remodeling, hyperplasia, and exaggerated
wound healing in bodily tissue (e.g., soft tissue, connective
tissue, bone, solid organs, blood vessel and the like). Examples of
diseases that are treatable or diagnosed by administering a
pharmaceutical composition comprising Q3 SPARC deletion mutant
polypeptide or anti-SPARC polypeptide antibody, such as, e.g.,
ant-Q3 SPARC deletion mutant polypeptide antibody therapy, include
cancer, diabetic or other retinopathy, inflammation, arthritis,
atherosclerosis, renal mesangial disease, restenosis in blood
vessels, artificial blood vessel grafts, or devices, and the
like.
[0119] The inventive method comprises administering to a mammal
afflicted with a disease characterized by over-expression of SPARC
a therapeutically effective amount of a pharmaceutical composition
comprising a chemotherapeutic agent or radioactive element coupled
to a Q3 SPARC deletion mutant polypeptide or an anti-SPARC
polypeptide, such as, e.g., an anti-Q3 SPARC deletion mutant
polypeptide antibody. The chemotherapeutic or radioactive agent can
be coupled to the antibody recognizing SPARC using any suitable
method. Preferably, the chemotherapeutic agent can be chemically
coupled to the compound via covalent bonds including, for example,
disulfide bonds.
[0120] For use in vivo, the Q3 SPARC deletion mutant polypeptide or
anti-SPARC polypeptide antibody, such as, e.g., anti-Q3 SPARC
deletion mutant polypeptide antibody, coupled or conjugated to a
therapeutic agent desirably is formulated into a pharmaceutical
composition comprising a physiologically acceptable carrier. Any
suitable physiologically acceptable carrier can be used within the
context of the invention, and such carriers are well known in the
art.
[0121] The carrier typically will be liquid, but also can be solid,
or a combination of liquid and solid components. The carrier
desirably is physiologically acceptable (e.g., a pharmaceutically
or pharmacologically acceptable) carrier (e.g., excipient or
diluent). Physiologically acceptable carriers are well known and
are readily available. The choice of carrier will be determined, at
least in part, by the location of the target tissue and/or cells,
and the particular method used to administer the composition.
[0122] Typically, such compositions can be prepared as injectables,
either as liquid solutions or suspensions; solid forms suitable for
using to prepare solutions or suspensions upon the addition of a
liquid prior to injection can also be prepared; and the
preparations can also be emulsified. The pharmaceutical
formulations suitable for injectable use include sterile aqueous
solutions or dispersions; formulations containing known protein
stabilizers and lyoprotectants, formulations including sesame oil,
peanut oil or aqueous propylene glycol, and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the formulation must be sterile and must
be fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. Solutions of the active compounds as free
base or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxycellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0123] The Q3 SPARC deletion mutant polypeptide or anti-SPARC, such
as, e.g., anti-Q3 SPARC deletion mutant polypeptide, therapy can be
formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the protein) and which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such as organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups also can be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0124] The composition can further comprise any other suitable
components, especially for enhancing the stability of the
composition and/or its end-use. Accordingly, there is a wide
variety of suitable formulations of the composition of the
invention. The following formulations and methods are merely
exemplary and are in no way limiting.
[0125] Formulations suitable for administration via inhalation
include aerosol formulations. The aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
can be formulated as non-pressurized preparations, for delivery
from a nebulizer or an atomizer.
[0126] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of a sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described. In a preferred embodiment of the
invention, the Q3 SPARC deletion mutant polypeptide or anti-SPARC,
such as, e.g., anti-Q3 SPARC deletion mutant polypeptide therapy is
formulated for injection (e.g., parenteral administration). In this
regard, the formulation desirably is suitable for intratumoral
administration, but also can be formulated for intravenous
injection, intraperitoneal injection, subcutaneous injection, and
the like.
[0127] Formulations suitable for anal administration can be
prepared as suppositories by mixing the active ingredient with a
variety of bases such as emulsifying bases or water-soluble bases.
Formulations suitable for vaginal administration can be presented
as pessaries, tampons, creams, gels, pastes, foams, or spray
formulas containing, in addition to the active ingredient, such
carriers as are known in the art to be appropriate.
[0128] In addition, the composition of the invention can comprise
additional therapeutic or biologically-active agents. For example,
therapeutic factors useful in the treatment of a particular
indication can be present. Factors that control inflammation, such
as ibuprofen or steroids, can be part of the composition to reduce
swelling and inflammation associated with in vivo administration of
the pharmaceutical composition and physiological distress.
[0129] The invention further provides for a method of classifying a
disease comprising detecting a SPARC polypeptide with a deletion of
the glutamine corresponding to amino acid position 20 in SEQ ID NO:
1. This classification can, for example, without limitation,
indicate the presence of a disease, provide information on the
stage of the disease, be informative of the prognosis for a given
patient or predict the response to therapy. Suitable diseases
include, without limitation, diseases involving neoangiogenesis
(e.g, cancer or retinopathies) or proliferative diseases (e.g.,
cancer, benign tumors, glomerulonephropathies, atherosclerosis, and
proliferative vascular restenosis). Suitable SPARC polypeptides
include, without limitation, wherein the mutant SPARC polypeptide
detected comprises the amino acid sequence of SEQ ID NO: 2.
[0130] Any suitable biological sample can be isolated from the
mammal in the context of the inventive method and used for
polypeptide and/or nucleic acid analysis. Preferably, the
biological sample is isolated from the tumor, such as by a tumor
biopsy. The biological sample is isolated from the mammal using
methods known in the art. Alternatively, the biological sample can
be isolated from a bodily fluid of the mammal, including, for
example, cerebrospinal fluid, blood, plasma, serum, or urine. In
particular, many protein purification techniques are known in the
art (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual,
Cold Spring Harbor, pp. 421-696 (1988)).
[0131] Any suitable method for the detection of a SPARC polypeptide
with a deletion of the glutamine at corresponding to amino acid
position 20 in SEQ ID NO: 1. can be used in accordance with the
invention including, but not limited to, the use of anti-Q3 SPARC
deletion mutant antibodies (e.g., Western blot, ELISA), the use of
SPARC-specific binding proteins (e.g., radiolabel SPARC ligands,
ELISA-like assays) or direct sequencing of partially purified or
purified SPARC polypeptides (e.g., Sanger sequencing, HPLC peptide
mapping and Edman degradation, mass spectrometry techniques,
enzymatic degradations) (see, e.g., Harlow & Lane, Antibodies,
A Laboratory Manual, Cold Spring Harbor, pp. 421-696 (1988);
Kirkpatrick et al., Nature Cell Biol. 7(8):750-7 (2005); Tomita et
al., J. Biol. Chem. 279(52):54161-72 (2004)).
[0132] The invention also provides for a method of classifying a
disease comprising detecting a method of classifying a disease
comprising detecting a nucleic acid encoding a SPARC polypeptide
with a deletion of the glutamine at the position corresponding to
amino acid position 20 of SEQ ID NO: 1, including wherein the
nucleic acid is DNA or RNA. Further, the invention provides for a
method of classifying a disease including, without limitation,
wherein the mutant SPARC nucleic acid detected comprises the
nucleic sequence of SEQ ID NO: 3. This classification can, for
example, without limitation, indicate the presence of a disease,
provide information on the stage of the disease, be informative of
the prognosis for a given patient or predict the response to
therapy. Suitable diseases include, without limitation, diseases
involving neoangiogenesis (e.g, cancer or retinopathies) or
proliferative diseases (e.g., cancer, benign tumors,
glomerulonephropathies, atherosclerosis, and proliferative vascular
restenosis).
[0133] Any suitable method can be using in accordance with the
invention for the detection of a nucleic acid encoding a SPARC
polypeptide with a deletion of the glutamine at the position
corresponding to amino acid position 20 of SEQ ID NO: 1.
[0134] Nucleic acid sequence information may be obtained from a
biological sample containing genetic material in numerous different
ways, particularly, nucleic acids (genetic material) containing the
sequence or sequences of interest. Many methods are known in the
art for extracting nucleic acids from biological samples. There are
many known methods for the separate or simultaneous isolation of
DNA and RNA from biological samples. Typically, DNA may be isolated
from a biological sample when first the sample is lysed and then
the DNA is isolated from the lysate according to any one of a
variety of multi-step protocols, which can take varying lengths of
time. DNA isolation methods may involve the use of phenol (see,
e.g., Sambrook & Russell, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, New York (2001), pp.
6.1-6.62). For example, a biological sample is lysed in a detergent
solution and the protein component of the lysate is digested with
proteinase for 12-18 hours. Next, the lysate is extracted with
phenol to remove most of the cellular components, and the remaining
aqueous phase is processed further to isolate DNA. In another
method, described in van Ness et al. (U.S. Pat. No. 5,130,423),
non-corrosive phenol derivatives are used for the isolation of
nucleic acids, resulting in a preparation that is a mix of RNA and
DNA.
[0135] Other methods for DNA isolation utilize non-corrosive
chaotropic agents. These methods, which are based on the use of
guanidine salts, urea and sodium iodide, involve lysis of a
biological sample in a chaotropic aqueous solution and subsequent
precipitation of the crude DNA fraction with a lower alcohol (see,
e.g., Analects, (1994) Vol 22, No. 4, Pharmacia Biotech; U.S. Pat.
No. 5,128,247). Numerous other methods are known in the art to
isolate both RNA and DNA, such as the one described by Chomczynski
(U.S. Pat. No. 5,945,515), whereby genetic material can be
extracted efficiently in as little as twenty minutes.
[0136] Once a subject's nucleic acid has been obtained from the
subject it may then be further analyzed to detect or determine the
presence or absence of one or more polymorphisms or mutations in
the sequence of interest, provided that the genetic material
obtained contains the sequence of interest. Particularly, a person
can detect a nucleic acid sequence encoding a SPARC polypeptide
comprising an amino acid sequence wherein the glutamine
corresponding to amino acid 20 in SEQ ID NO: 1 is deleted. The
sequence of interest may also include other mutations, or may also
contain some of the sequence surrounding the mutation of
interest.
[0137] Detection or determination of a nucleotide identity, or the
presence of one or more single nucleotide polymorphism(s) (SNP
typing), may be accomplished by any one of a number methods or
assays known in the art. Many DNA typing methodologies are useful
detection of SNPs including but, not limited to, sequence-specific
hybridization, primer extension, oligonucleotide ligation, and
invasive cleavage methods. Suitable reactions can occur in solution
or on a solid support such as a glass slide, a chip, a bead, and
the like.
[0138] Sequence-specific hybridization involves a hybridization
probe, which is capable of distinguishing between two DNA targets
differing at one nucleotide position by hybridization. In one
approach, "Molecular beacons," the hybridization probe has 3' and
5' reporter and quencher molecules and 3' and 5' sequences which
are complementary such that absent an adequate binding target for
the intervening sequence the probe will form a hairpin loop. The
hairpin loop keeps the reporter and quencher in close proximity
resulting in quenching of the fluorophor which reduces fluorescence
emissions. Upon hybridization to the target the fluorophor and the
quencher are sufficiently separated to allow fluorescence to be
emitted from the fluorophor. Primer extension reactions (i.e. mini
sequencing, nucleotide-specific extensions, or simple PCR
amplification) can also be useful in sequence discrimination
reactions.
[0139] Oligonucleotide ligation assays require two
sequence-specific probes and one common ligation probe per SNP. The
common ligation probe hybridizes adjacent to a sequence-specific
probe and when there is a perfect match of the appropriate
sequence-specific probe, the ligase joins both the
sequence-specific and the common probes.
[0140] Invasive cleavage method requires an oligonucleotide called
an "Invader.RTM." (Third Wave Technologies) probe and
sequence-specific probes to anneal to the target DNA with an
overlap of one nucleotide. When the sequence-specific probe is
complementary to the polymorphic base, overlaps of the 3' end of
the invader oligonucleotide form a structure that is recognized and
cleaved by a Flap endonuclease releasing the 5' arm of the allele
specific probe.
[0141] 5' exonuclease activity or "TaqMan.RTM." assay (Applied
Biosystems) is based on the 5' nuclease activity of Taq polymerase
that displaces and cleaves the oligonucleotide probes hybridized to
the target DNA generating a fluorescent signal. It is necessary to
have two probes that differ at the polymorphic site wherein one
probe is complementary to the `normal` sequence and the other to
the mutation of interest. These probes have different fluorescent
dyes attached to the 5' end and a quencher attached to the 3' end
when the probes are intact the quencher interacts with the
fluorophor by fluorescence resonance energy transfer (FRET) to
quench the fluorescence of the probe. During the PCR annealing step
the hybridization probes hybridize to target DNA. In the extension
step the 5' fluorescent dye is cleaved by the 5' nuclease activity
of Taq polymerase, leading to an increase in fluorescence of the
reporter dye. Mismatched probes are displaced without
fragmentation. The presence of a mutation in a sample is determined
by measuring the signal intensity of the two different dyes.
[0142] In addition, there are numerous other methods for sequence
discrimination and detection such as arrayed primer extension mini
sequencing, tag microarrays and sequence-specific extension could
be performed on a microarray. One such array based genotyping
platform is the microsphere based the "tag-it" high throughput
genotyping array (Bortolin et al., Clinical Chemistry 50(11):
2028-36 (2004)).
[0143] Mutation detection methods can also include but, are not
limited to the following:
[0144] A Restriction Fragment Length Polymorphism (RFLP)
strategy--An RFLP gel-based analysis can be used to indicate the
presence or absence of a specific mutation at polymorphic sites
within a gene based on the presence or absence of a restriction
site;
[0145] Sequencing--For example, the Maxam-Gilbert technique for
sequencing (Maxam & Gilbert, Proc. Natl. Acad. Sci. USA
74(4):560-564 (1977)) or the dideoxy method of sequencing was
published by Sanger et al. (Proc. Natl. Acad. Sci. USA
74(12):5463-5467 (1977)). In addition, RNA sequencing methods are
also known. For example, reverse transcriptase with
dideoxynucleotides have been used to sequence encephalomyocarditis
virus RNA (Zimmern & Kaesberg, Proc. Natl. Acad. Sci. USA
75(9):4257-4261 (1978)). Mills and Kramer (Proc. Natl. Acad. Sci.
USA (1979) 76(5):2232-2235) describe the use of Q.beta. replicase
and the nucleotide analog inosine for sequencing RNA in a
chain-termination mechanism. Direct chemical methods for sequencing
RNA are also known (e.g., Peattie, Proc. Natl. Acad. Sci. USA
76(4):1760-1764 (1979)). Nucleic acid sequences can also be read by
stimulating the natural fluoresce of a cleaved nucleotide with a
laser while the single nucleotide is contained in a fluorescence
enhancing matrix (U.S. Pat. No. 5,674,743);
[0146] Hybridization methods for the identification of single
nucleotide polymorphisms or other mutations involving a few
nucleotides are described in the U.S. Pat. Nos. 6,270,961 &
6,025,136;
[0147] A template-directed dye-terminator incorporation with
fluorescent polarization-detection (TDI-FP) method is described by
Freeman et al. (J. Mol. Diagnostics. 4(4):209-215 (2002));
[0148] Oligonucleotide ligation assay (OLA) is based on ligation of
probe and detector oligonucleotides annealed to a polymerase chain
reaction amplicon strand with detection by an enzyme immunoassay
(see e.g., Villahermosa, J. Hum. Virol. 4(5):238-48 (2001));
[0149] Ligation-Rolling Circle Amplification (L-RCA) has also been
successfully used for genotyping single nucleotide polymorphisms as
described by Qi et al. (Nucleic Acids Res. 29(22):E116 (2001));
[0150] 5' nuclease assay has also been successfully used for
genotyping single nucleotide polymorphisms (Aydin et al.,
Biotechniques (4):920-2, 924, 926-8 (2001));
[0151] Polymerase proofreading methods are used to determine SNPs
identities, as described in PCT Publication WO 01/81631;
[0152] Detection of single base pair DNA mutations by
enzyme-amplified electronic transduction is described in Patolsky
et al., Nature Biotech. (2001) 19(3):253-257 (2001);
[0153] Gene chip technologies are also known for single nucleotide
polymorphism discrimination whereby numerous polymorphisms may be
tested for simultaneously on a single array (See e.g., Gilles et
al. Nat. Biotechnology 17(4):365-70 (1999));
[0154] Matrix Assisted Laser Desorption Ionization-Time Of Flight
(MALDI-TOF) mass spectroscopy is also useful in the genotyping
single nucleotide polymorphisms through the analysis of
microsequencing products (See, e.g., Haff & Smirnov, Nucleic
Acids Res. 25(18):3749-50 (1997)).
[0155] Sequence-specific PCR methods have also been successfully
used for genotyping single nucleotide polymorphisms (Hawkins et
al., Hum. Mutat. (2002) 19(5):543-553). The invention, therefore,
provides for a method, wherein the nucleic acid encoding a Q3 SPARC
deletion mutant polypeptide is detected using a method comprising
the polymerase chain reaction (PCR). Further, the invention
provides for a method, wherein mutant and non-mutant SPARC nucleic
acids are detected using a method comprising PCR, wherein one or
more forward primers selected from primers comprising the sequences
of SEQ ID NOS: 7-9 and a reverse primer comprising the sequence of
SEQ ID NO: 10.
[0156] Alternatively, if a subject's genetic sequence data is
already known, then obtaining may involve retrieval of the
subject's nucleic acid sequence data from a database, followed by
determining or detecting the identity of a nucleic acid or genotype
at a polymorphism site by reading the subject's nucleic acid
sequence at the polymorphic site.
[0157] The invention also provides for a means of transporting the
therapeutic composition across the endothelial barrier from the
blood vessel into the tumor interstitium. The main hurdle in
antibody therapy and chemotherapy is the translocation across the
endothelial barrier into tumor interstitium. Albumin utilizes the
albumin receptor transport mechanism to cross the endothelial
barrier. This transport mechanism may be the same as those reported
by the literature (gp60 and albondin) or by other mechanisms. The
therapeutic agent piggy backed onto albumin exhibited enhanced
tumoral uptake (Example-ABI-007 tumor uptake and transcytosis).
Enhanced translocation across the endothelial barrier can be
achieved using the physiological albumin transport mechanism.
[0158] For small molecules, modifications can be made so that the
drug affinity for albumin is increased. For formulations of small
molecules, solvent which prevents the binding of the drug to
albumin can be removed. Alternatively, the small molecule could
linked to albumin, antibody against albumin, fragments thereof or
ligands for albumin-receptor such as described below.
[0159] For biologic molecules such as proteins, antibodies and
fragments, it is possible to engineer the biologics with an albumin
binding peptide such that the biologics will exhibit affinity for
albumin. The peptide can either be albumin binding sequence,
antibody or antibody fragment against albumin, antibody or antibody
fragment against albumin carriers such as gp60/albondin/scavenger
receptor/or TGF-beta receptor, or antibody to any of the proteins
found in the caveolae--the transporter of albumin. The invention
also contemplates an antibody or suitable fragment thereof prepared
as a chimera with one valence for SPARC and another valence for an
effector of transendothelial transport such as
gp60/albondin/scavenger receptor/or TGF-beta receptor, or against
any of the proteins found in the caveolae of the endothelial
cell.
[0160] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0161] This example illustrates the co-localization of SPARC with
albumin in an MX-1 tumor xenograft.
[0162] Paclitaxel albumin nanoparticles (Abraxane, ABX or ABI-007)
have been shown to have an improved response rate over Taxol (TAX)
in a Phase 3 metastatic breast cancer trial (33% vs. 19%,
p<0.0001) (see, e.g., O'Shaughnessy, SABCS 2003).
Albumin-mediated transendothelial transport of paclitaxel (P) and
increased intratumoral accumulation of paclitaxel for ABX versus
TAX was demonstrated recently (see, e.g., Desai, SABCS 2003).
Albumin binds to SPARC (see, e.g., Schnitzer, J. Biol. Chem.
269:6072-82 (1994)).
[0163] The MX-1 tumor cell line is derived from a human breast
cancer. Serial cryosections of human MX-1 tumor xenograft, human
primary breast tumor tissues (n=141), and normal human breast
tissue (n=115) were immunostained and scored (0-4) for albumin,
SPARC (using anti-SPARC antibody), and caveolin-1 staining.
Cultured MX-1 cells also were immunostained for SPARC. Paclitaxel
albumin nanoparticles (Abraxane, ABX or ABI-007) and Taxol (TAX)
were prepared with radioactive paclitaxel (P) (20 mg/kg IV), and
were used to determine the biodistribution of paclitaxel in normal
tissues of athymic mice.
[0164] Albumin staining in the MX-1 tumor was focal and
co-localized with SPARC (FIG. 1). Caveolin-1 staining confirmed
that blood vessel density in albumin-containing areas was no
different from albumin-free areas. SPARC expression by MX-1
cultured cells was confirmed by positive staining with anti-SPARC
antibody. Paclitaxel accumulation in normal tissues (SPARC
negative) was significantly lower for ABX as compared to TAX
(p<0.004) for 7/10 tissues. 46% of the human primary breast
tumors exhibited strong SPARC staining (score>2), as compared to
1% for normal tissues (p<0.0001). In a subset of 50 tumor
tissues, SPARC expression did not correlate with staging, ER
status, or PgR status; however, there was trend for high SPARC
expression among p53-negative tumors.
[0165] The co-localization of albumin and SPARC suggests that
SPARC, by its albumin binding activity, may behave as an
intratumoral target for albumin binding in breast tumors. As
transport of paclitaxel in ABX is dependent on albumin (see, e.g.,
Desai SABCS, 2003), this may explain the improved tumor
accumulation of ABX as compared to TAX. ABX accumulation in normal
tissues was lower than for TAX, consistent with lack of SPARC
expression in normal tissues. Screening of patients for SPARC
allows for the identification of patients more responsive to ABX.
The presence of SPARC in these tumors allows for targeting and
therapy using anti-SPARC antibody.
Example 2
[0166] This example illustrates endothelial receptor
(gp60)-mediated caveolar transcytosis of paclitaxel albumin
nanoparticles (ABI-007).
[0167] Paclitaxel (P) albumin nanoparticles (Abraxane, ABX or
ABI-007) demonstrated improved response rate over Taxol in a phase
III metastatic breast cancer trial (33% vs 19%, p<0.0001)
(SABCS, O'Shaughnessy et al, 2003). Cremophor in Taxol (TAX)
entraps P in micelles in plasma, reducing the paclitaxel available
for cellular partitioning (see, e.g., Sparreboom et al., Cancer.
Res., 59, 1454 (1999)). Studies in athymic mice have shown 30-40%
higher intratumor paclitaxel concentrations with ABX as compared to
equal doses of TAX (SABCS, Desai et al, 2003). Albumin is
transported across endothelial cells (EC) by specific receptor
(gp60)-mediated caveolar transport (see, e.g., John et al., Am. J.
Physiol., 284, L187 (2001)). It was hypothesized that albumin-bound
paclitaxel in ABX may be transported across tumor microvessel EC by
gp60, and this mechanism may be particularly active for ABX as
compared to TAX.
[0168] A series of experiments were performed to evaluate binding
and transport of paclitaxel by human umbilical vein endothelial
cells (HUVEC) and human lung microvessel endothelial cells (HLMVEC)
for ABX and TAX. Fluorescent paclitaxel (FP) was used as a probe
and fluorescent ABX and TAX were formulated with FP to probe the
binding and transport of paclitaxel across EC monolayers grown on a
transwell apparatus.
[0169] Binding of paclitaxel to cells (HUVEC) was 10.times. higher
for ABX than TAX. The transport of paclitaxel from ABX across EC
monolayers was enhanced by 2-3 fold and 2-4 fold for HUVEC and
HMVEC, respectively, as compared to TAX. Transport was dependent on
albumin. Transport of paclitaxel from ABX was inhibited by the
presence of anti-SPARC antibody, which is known to bind gp60, the
receptor required for caveolar albumin transcytosis. Known
inhibitors of calveolar transcytosis, NEM and (3-methyl
cyclodextrin (BMC), also inhibited the transport of paclitaxel from
ABX across the endothelial monolayers (FIG. 2). Inhibition of
caveolar transport decreased transport of P from ABX to the level
of TAX transport.
[0170] These results demonstrate that paclitaxel from ABX is
actively transported across EC by gp60-mediated caveolar
transcytosis, whereas P from TAX appears to be transported at a 2-4
fold lower rate primarily by a paracellular (non-caveolar)
mechanism. This pathway may in part be responsible for increased
intratumoral concentrations of paclitaxel seen for ABX relative to
TAX. Cremophor in TAX inhibits transcytosis of paclitaxel across
endothelial cells.
Example 3
[0171] This example illustrates the expression of surface SPARC in
MX-1 tumor cells.
[0172] MX-1 cells were cultured on a coverslip and stained with an
antibody directed against human SPARC using methods known in the
art. Antibody staining was observed, which demonstrates that MX-1
is expressing SPARC. These results suggest that SPARC expression
detected in MX-1 tumor cells is a result of SPARC secretion by MX-1
tumor cells. Staining was more intense for MX-1 tumor cells than
that of normal primary cells such as HUVEC (human umbilical vein
endothelial cells), HLMVEC (Human lung microvessel endothelial
cells), and HMEC (Human mammary epithelial cells). Although the
majority of the SPARC staining was internal SPARC, significant
level of surface SPARC was detected as demonstrated by confocal
microscopy and staining of unpermeabilized cells.
Example 4
[0173] This example illustrates the internalization of labeled
albumin into MX-1 tumor cells and colocalization within the MX-1
cell with intracellular SPARC expression.
[0174] MX-1 cells were cultured on a coverslip and permeabilized
with suitable agents. Cells were exposed to fluorescent albumin and
following washing were exposed to SPARC antibody. This was followed
by exposure to a secondary antibodies having a different
fluorescent tag than the albumin. It was surprisingly observed that
the labeled albumin colocalised with the presence of SPARC within
the cell indicating that albumin was rapidly internalized and
targeted intracellular SPARC.
Example 5
[0175] This example demonstrates an increase in endothelial
transcytosis via gp60 (albumin receptor) of pharmaceutical
compositions comprising paclitaxel and albumin as compared to
Taxol.
[0176] Human lung microvessel endothelial cells (HLMVEC) were grown
to confluence on a transwell. The inventive pharmaceutical
composition comprising paclitaxel and albumin, or Taxol containing
fluorescent paclitaxel (Flutax) at a concentration of 20 .mu.g/mL,
was added to the upper transwell chamber.
[0177] The transport of paclitaxel by transcytosis from the upper
chamber to the lower chamber was monitored continuously using a
fluorometer. A control containing only Flutax without albumin was
also used. The control with Flutax showed no transport, validating
the integrity of the confluent HLMVEC monolayer. Transport of
paclitaxel from the albumin-paclitaxel composition was much faster
than paclitaxel from Taxol in the presence of 5% HSA (physiological
concentration). Transport rate constants (K.sub.t) for the
albumin-paclitaxel composition and Taxol were 1.396 h.sup.-1 and
0.03 h.sup.-1, respectively. The total amount of paclitaxel
transported across the monolayer was three times higher for the
albumin-paclitaxel composition than Taxol. Thus, the use of albumin
or other suitable mimetic including antibodies or fragments against
the gp60 receptor or other endothelial cell receptor can assist in
the transport of a desired therapeutic agent across the endothelial
barrier into the tumor interstitium.
Example 6
[0178] This example illustrates the overexpression of SPARC protein
in human breast carcinoma cells.
[0179] SPARC expression in human breast carcinoma cells was
determined using a tumor array from Cybrdi, Inc. (Gaithersburg,
Md.). The results of this analysis are set forth in Table 1.
Intensity of staining was scored from "Negative" to 4+, with the
higher number corresponding to greater intensity of overexpression.
49% of breast carcinoma stained positive (2+ and above) for SPARC,
as compared to 1% of normal tissue (p<0.0001).
TABLE-US-00001 TABLE 1 SPARC Staining (%) Negative -/+ 1+ 2+ 3+ 4+
Carcinoma Cells 31 (34%) 14 (15%) 1 (1%) 11 (12%) 9 (10%) 25 (27%)
Normal Cells 93 (89%) 7 (7%) 4 (4%) 1 (1%) 0 (0%) 0 (0%)
Example 7
[0180] This example demonstrate the specific binding of anti-SPARC
antibody to SPARC.
[0181] Whole cell extract was prepared from HUVEC cells by
sonication. The protein was separated by 5-15% SDS-PAGE,
transferred onto PVDF membrane and visualized with a polyclonal
antibody against SPARC and a monoclonal antibody against SPARC.
Both antibodies reacted to a single band at 38 kDa, the correct
molecular weight for SPARC. When MX-1 was analyzed by the same
method, SPARC was detected in both the clarified cell lysate or the
membrane rich membrane fraction.
Example 8
[0182] This example demonstrates correlation of SPARC
overexpression with high response rates using nanoparticle
albumin-bound paclitaxel (ABI-007) in squamous head and neck
cancers.
[0183] In phase I and II clinical studies of patients with squamous
cell carcinoma (SCC) of head and neck (H&N) and anal canal,
response rates of 78% and 64% were observed, respectively, for
intra-arterially delivered Nanoparticle Albumin-Bound Paclitaxel
(Abraxane, ABX or ABI-007) (see, e.g., Damascelli et al., Cancer,
92(10), 2592-2602 (2001), and Damascelli et al., AJR, 181, 253-260
(2003)). In comparing in vitro cytoxicity of ABX and Taxol (TAX),
we observed that a squamous cervix (A431) line demonstrated
improved IC.sub.50s for ABX (0.004 .mu.g/ml) vs TAX (0.012
.mu.g/ml). Albumin-mediated transendothelial caveolar transport of
paclitaxel (P) and increased intratumoral accumulation of P for ABX
vs TAX was demonstrated recently (see, e.g., Desai, SABCS
2003).
Human H&N tumor tissues (n=119) and normal human H&N tissue
(n=15) were immunostained and scored (0-4) for SPARC staining using
a tumor and normal tissue array. Immunostaining was performed using
polyclonal rabbit anti-SPARC antibody. SPARC was overexpressed
(score>2) in 60% (72/119) of the H&N tumors versus 0% (0/15)
in normal tissues (p<0.0001). This may explain the high
single-agent activity of ABX seen previously in squamous H&N
cancers due to binding of albumin-bound paclitaxel to SPARC
expressed in these tumors.
[0184] In a new phase I dose escalation study (ABX given IV over 30
minutes q3w), a subset of head and neck cancer patients (n=3) were
analyzed for response to ABX. In the phase I study, 2/3H&N
patients achieved partial response (PR) after 2 cycles of treatment
at dose levels of 135 mg/m.sup.2 (1 pt) and 225 mg/m.sup.2 (1 pt).
A third patient at 260 mg/m.sup.2 progressed. Tumor tissues from
these patients were stained for SPARC and 1 responding patients
showed strong overexpression for SPARC.
[0185] In another phase II clinical study of patients with squamous
cell carcinoma (SCC) of head and neck (H&N) treated with
intra-arterial Abraxane, an overall response rate of 68% was noted.
In 10 responding patients whose tissues were analyzed for SPARC
staining, 70% were found to strongly overexpress SPARC.
Example 9
[0186] This example demonstrates the cloning and sequencing of a
human SPARC cDNA which encodes a Q3 SPARC deletion mutant
polypeptide.
[0187] A normal human prostate cDNA library was screened for clones
containing SPARC cDNAs using a high throughput screening method as
described in U.S. Pat. No. 6,316,193. A SPARC cDNA clone was
isolated and sequenced using primers distributed along its length
to obtain the complete cDNA sequence (see FIG. 3). Sequencing data
were assembled using the Vector NTI program. The reference Genebank
sequence for human SPARC cDNA (NM 003118 [gi:48675809], last
updated on Dec. 4, 2005) was used for the assembly. The
distribution of the sequencing data for each primer is shown in
FIG. 3 and the results of the individual sequence reactions in FIG.
4.
[0188] The complete assembled 2,956 base pair cDNA sequence, SEQ ID
NO: 3, is shown in FIG. 5. The bold/underlined trinucleotides in
FIG. 5 are, respectively, the translational start site for the
encoded Q3 SPARC deletion mutant polypeptide and the first codon in
the encoded mature Q3 SPARC deletion mutant polypeptide (the amino
acid sequence after the 17 amino acid leader sequence). The SEQ ID
NO: 3 open reading frame encodes a full length SPARC protein with a
deletion of the glutamine corresponding to the glutamine at
position 20 in the wild type SPARC amino acid sequence (SEQ ID NO:
1).
[0189] The translation of the full length open reading frame
encoding the Q3 SPARC deletion mutant polypeptide from SEQ ID NO: 3
is shown in FIG. 6. The start codon of this open reading frame is
at nucleotide 87 of SEQ ID NO: 3 and the open reading frame ends at
nucleotide 992 of SEQ ID NO: 3. The translated amino acid sequence
is 302 codons long and is disclosed by SEQ ID NO: 4. The DNA
sequence of the full length open reading frame is disclosed by SEQ
ID NO: 5.
[0190] FIG. 7 shows the amino acid sequence of the mature Q3 SPARC
deletion mutant polypeptide (the sequence without the 17 amino acid
leader sequence) encoded by SEQ ID NO: 3. This results from a
translation of nucleotides 138 through 992 of SEQ ID NO: 3. The
mature Q3 SPARC deletion mutant polypeptide is SEQ ID NO: 2 and the
DNA sequence encoding the mature Q3 SPARC deletion mutant
polypeptide is SEQ ID NO: 6.
Example 10
[0191] This example demonstrates the identification of the deletion
of the glutamine at position 20 in the wild type SPARC amino acid
sequence (SEQ ID NO: 1) in the SPARC polypeptide encoded by SEQ ID
NO: 3 (a "Q3 SPARC deletion").
[0192] Alignment of SEQ ID NO: 3 DNA sequence with the human SPARC
cDNA sequence deposited in Genebank (FIG. 8) demonstrated that SEQ
ID NO: 3 contains a 3 base pair deletion (boldface) corresponding
to the codon encoding the Q3 of the mature SPARC protein. In
addition, alignment of the translated SPARC open reading from SEQ
ID NO: 3, SEQ ID NO: 4, demonstrated deletion of the glutamine at
position 20 in the wild type SPARC amino acid sequence (the Q3
amino acid) (FIG. 9).
Example 11
[0193] This example demonstrates the evolutionary conservation of
the glutamine residues at amino acid positions 3 and 4 of the
mature SPARC protein (the "Q3Q4" amino acids).
[0194] Examination of the amino acid sequence for SPARC across the
phylogenetic tree revealed the conservation of Q3Q4 across mammal
and amphibian. A single glutamine was only observed in rat and
chicken. Neither Q3 nor Q3Q4 was observed in fish. The skeletal
structures of fish and bird are much lighter than that of mammal
and amphibian, suggesting that this site for tranglutaminase is
involved in the formation of highly crosslinked and structurally
compact skeletal structure. SPARC with only Q3 could be free to
participate in non-skeletal functions such as angiogenesis, tumor
growth, albumin binding, and the like.
[0195] The conservation of the Q3Q4 pair of glutamines in higher
organisms suggests that a Q3 SPARC deletion mutant polypeptide
could have novel properties.
Example 12
[0196] This example demonstrates the subcloning of a Q3 SPARC
deletion mutant polypeptide encoding cDNA into an expression
vector.
[0197] SPARC cDNA was excised with Not I and subcloned into the Not
I site of pcDNA3.1 to give rise to pVT1000Q3. The orientation of
the insert relative to CMV promoter was confirmed by restriction
digest (Not I, Eco RV, Eco RI, Ssp I) and sequencing. Restriction
digestion gave rise to fragments with the predicted size for all
four restriction enzymes (FIG. 11). The restriction map for the
pVT1000Q3 construct is shown in FIG. 12.
[0198] The inventors expect that pVT1000Q3 will readily transfect
the CHO, MDA-MB-231, PC3, HT29, and MX-1 cell lines using
transfection methods well known to those of ordinary skill in the
art.
Example 13
[0199] This example demonstrates primer directed mutagenesis by the
insertion of the Q3 into pVT1000Q3 generating a sequence encoding a
wild type SPARC polypeptide.
[0200] A polymerase chain reaction (PCR) primer containing the Q3
codon (see FIG. 13) was used in primer directed mutagenesis method
well known to those of ordinary skill in the art. The resulting
plasmid, pVT1000 wt, exhibited the expected wild type Q3Q4 SPARC
sequence.
Example 14
[0201] This example demonstrates the PCR detection of a nucleic
acid encoding a Q3 SPARC deletion mutant polypeptide, i.e., the "Q3
mutation."
[0202] The location and sequence of primers designed to detect the
Q3 mutation and the wild type form of SPARC cDNA are shown in FIG.
14. The inventive PCR primers SPARC-FQ3, SPARC-FWT, and
SPARC-FQ3&W (SEQ ID NOS: 7-9, respectively) as forward primers
and inventive molecule SPARC-RQ3&W (SEQ ID NO: 10) as an
inventive reverse PCR primer were used in a standard reverse
transcription PCR (RT-PCR) reaction well known to those of ordinary
skill in the art. The Q3 mutation and wild type sequences were
distinguished using the inventive primers in a PCR reaction
followed by gel electrophoresis as shown in Table 2.
TABLE-US-00002 TABLE 2 Reaction Results for wild Mix Primers
Results for Q3 mutant type #1 FQ3 + RQ3&WT 309 base pair band
No products #2 FQ3 + FQ3&W + RQ3&WT 309 + 222 base pair
bands 222 base pair band #3 FW + RQ3&WT No products 309 base
pair band #4 FW3 + FQ3&WT + RQ3&WT 222 base pair band 309 +
222 base pair bands
[0203] The PCR amplification conditions used were: a hot start of
95.degree. C. for 10 minutes, followed by 30 cycles of 95.degree.
C. for 30 Sec., 62.degree. C. for 30 Sec., 72.degree. C. for 40
Sec., and final extension at 72.degree. C. for 10 minutes.
pVT1000Q3, which carries the Q3 mutation, and pVT1000 wt, which
carries the wild type SPARC, are used as controls. The Q3 mutation
was detected by the absence of the 309 base pair product with
reaction mixes #3 and #4 and the presence of the 309 base pair
product with reaction mixes #1 and #2. The wild type sequence was
detected as presence of the 309 base pair product with reaction
mixes #3 and #4 and the absence of the 309 base pair product with
reaction mix #2. With reaction mix #1, the wild type sequence
yielded a 309 base pair product but, at much weaker level than with
reaction mixes #3 and #4. Therefore, reaction mix #3 is more robust
for detecting wild type SPARC sequences (non-Q3 mutant sequences)
(FIG. 15).
[0204] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0205] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0206] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
241303PRTHomo sapiens 1Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu
Ala Gly Arg Ala Leu1 5 10 15Ala Ala Pro Gln Gln Glu Ala Leu Pro Asp
Glu Thr Glu Val Val Glu 20 25 30Glu Thr Val Ala Glu Val Thr Glu Val
Ser Val Gly Ala Asn Pro Val 35 40 45Gln Val Glu Val Gly Glu Phe Asp
Asp Gly Ala Glu Glu Thr Glu Glu 50 55 60Glu Val Val Ala Glu Asn Pro
Cys Gln Asn His His Cys Lys His Gly65 70 75 80Lys Val Cys Glu Leu
Asp Glu Asn Asn Thr Pro Met Cys Val Cys Gln 85 90 95Asp Pro Thr Ser
Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val Cys 100 105 110Ser Asn
Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala Thr 115 120
125Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu Asp
130 135 140Tyr Ile Gly Pro Cys Lys Tyr Ile Pro Pro Cys Leu Asp Ser
Glu Leu145 150 155 160Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu
Lys Asn Val Leu Val 165 170 175Thr Leu Tyr Glu Arg Asp Glu Asp Asn
Asn Leu Leu Thr Glu Lys Gln 180 185 190Lys Leu Arg Val Lys Lys Ile
His Glu Asn Glu Lys Arg Leu Glu Ala 195 200 205Gly Asp His Pro Val
Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn Tyr 210 215 220Asn Met Tyr
Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp Gln225 230 235
240His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu Arg
245 250 255Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe
Glu Thr 260 265 270Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Asp
Glu Trp Ala Gly 275 280 285Cys Phe Gly Ile Lys Gln Lys Asp Ile Asp
Lys Asp Leu Val Ile 290 295 3002285PRTHomo sapiens 2Ala Pro Gln Glu
Ala Leu Pro Asp Glu Thr Glu Val Val Glu Glu Thr1 5 10 15Val Ala Glu
Val Thr Glu Val Ser Val Gly Ala Asn Pro Val Gln Val 20 25 30Glu Val
Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr Glu Glu Glu Val 35 40 45Val
Ala Glu Asn Pro Cys Gln Asn His His Cys Lys His Gly Lys Val 50 55
60Cys Glu Leu Asp Glu Asn Asn Thr Pro Met Cys Val Cys Gln Asp Pro65
70 75 80Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val Cys Ser
Asn 85 90 95Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala Thr
Lys Cys 100 105 110Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His
Leu Asp Tyr Ile 115 120 125Gly Pro Cys Lys Tyr Ile Pro Pro Cys Leu
Asp Ser Glu Leu Thr Glu 130 135 140Phe Pro Leu Arg Met Arg Asp Trp
Leu Lys Asn Val Leu Val Thr Leu145 150 155 160Tyr Glu Arg Asp Glu
Asp Asn Asn Leu Leu Thr Glu Lys Gln Lys Leu 165 170 175Arg Val Lys
Lys Ile His Glu Asn Glu Lys Arg Leu Glu Ala Gly Asp 180 185 190His
Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn Tyr Asn Met 195 200
205Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp Gln His Pro
210 215 220Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu Arg
Ala Pro225 230 235 240Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe
Phe Glu Thr Cys Asp 245 250 255Leu Asp Asn Asp Lys Tyr Ile Ala Leu
Asp Glu Trp Ala Gly Cys Phe 260 265 270Gly Ile Lys Gln Lys Asp Ile
Asp Lys Asp Leu Val Ile 275 280 28532956DNAHomo sapiens 3ctccacattc
ccgcggtcct tcagactgcc cggagagcgc gctctgcctg ccgcctgcct 60gcctgccact
gagggttccc agcaccatga gggcctggat cttctttctc ctttgcctgg
120ccgggagggc cttggcagcc cctcaagaag ccctgcctga tgagacagag
gtggtggaag 180aaactgtggc agaggtgact gaggtatctg tgggagctaa
tcctgtccag gtggaagtag 240gagaatttga tgatggtgca gaggaaaccg
aagaggaggt ggtggcggaa aatccctgcc 300agaaccacca ctgcaaacac
ggcaaggtgt gcgagctgga tgagaacaac acccccatgt 360gcgtgtgcca
ggaccccacc agctgcccag cccccattgg cgagtttgag aaggtgtgca
420gcaatgacaa caagaccttc gactcttcct gccacttctt tgccacaaag
tgcaccctgg 480agggcaccaa gaagggccac aagctccacc tggactacat
cgggccttgc aaatacatcc 540ccccttgcct ggactctgag ctgaccgaat
tccccctgcg catgcgggac tggctcaaga 600acgtcctggt caccctgtat
gagagggatg aggacaacaa ccttctgact gagaagcaga 660agctgcgggt
gaagaagatc catgagaatg agaagcgcct ggaggcagga gaccaccccg
720tggagctgct ggcccgggac ttcgagaaga actataacat gtacatcttc
cctgtacact 780ggcagttcgg ccagctggac cagcacccca ttgacgggta
cctctcccac accgagctgg 840ctccactgcg tgctcccctc atccccatgg
agcattgcac cacccgcttt ttcgagacct 900gtgacctgga caatgacaag
tacatcgccc tggatgagtg ggccggctgc ttcggcatca 960agcagaagga
tatcgacaag gatcttgtga tctaaatcca ctccttccac agtaccggat
1020tctctcttta accctcccct tcgtgtttcc cccaatgttt aaaatgtttg
gatggtttgt 1080tgttctgcct ggagacaagg tgctaacata gatttaagtg
aatacattaa cggtgctaaa 1140aatgaaaatt ctaacccaag acatgacatt
cttagctgta acttaactat taaggccttt 1200tccacacgca ttaatagtcc
catttttctc ttgccatttg tagctttgcc cattgtctta 1260ttggcacatg
ggtggacacg gatctgctgg gctctgcctt aaacacacat tgcagcttca
1320acttttctct ttagtgttct gtttgaaact aatacttacc gagtcagact
ttgtgttcat 1380ttcatttcag ggtcttggct gcctgtgggc ttccccaggt
ggcctggagg tgggcaaagg 1440gaagtaacag acacacgatg ttgtcaagga
tggttttggg actagaggct cagtggtggg 1500agagatccct gcagaaccca
ccaaccagaa cgtggtttgc ctgaggctgt aactgagaga 1560aagattctgg
ggctgtgtta tgaaaatata gacattctca cataagccca gttcatcacc
1620atttcctcct ttacctttca gtgcagtttc ttttcacatt aggctgttgg
ttcaaacttt 1680tgggagcacg gactgtcagt tctctgggaa gtggtcagcg
catcctgcag ggcttctcct 1740cctctgtctt ttggagaacc agggctcttc
tcaggggctc tagggactgc caggctgttt 1800cagccaggaa ggccaaaatc
aagagtgaga tgtagaaagt tgtaaaatag aaaaagtgga 1860gttggtgaat
cggttgttct ttcctcacat ttggatgatt gtcataaggt ttttagcatg
1920ttcctccttt tcttcaccct cccctttttt cttctattaa tcaagagaaa
cttcaaagtt 1980aatgggatgg tcggatctca caggctgaga actcgttcac
ctccaagcat ttcatgaaaa 2040agctgcttct tattaatcat acaaactctc
accatgatgt gaagagtttc acaaatcctt 2100caaaataaaa agtaatgact
tagaaactgc cttcctgggt gatttgcatg tgtcttagtc 2160ttagtcacct
tattatcctg acacaaaaac acatgagcat acatgtctac acatgactac
2220acaaatgcaa acctttgcaa acacattatg cttttgcaca cacacacctg
tacacacaca 2280ccggcatgtt tatacacagg gagtgtatgg ttcctgtaag
cactaagtta gctgttttca 2340tttaatgacc tgtggtttaa cccttttgat
cactaccacc attatcagca ccagactgag 2400cagctatatc cttttattaa
tcatggtcat tcattcattc attcattcac aaaatattta 2460tgatgtattt
actctgcacc aggtcccatg ccaagcactg gggacacagt tatggcaaag
2520tagacaaagc atttgttcat ttggagctta gagtccagga ggaatacatt
agataatgac 2580acaatcaaat ataaattgca agatgtcaca ggtgtgatga
agggagagta ggagagacca 2640tgagtatgtg taacaggagg acacagcatt
attctagtgc tgtactgttc cgtacggcag 2700ccactaccca catgtaactt
tttaagattt aaatttaaat tagttaacat tcaaaacgca 2760gctccccaat
cacactagca acatttcaag tgcttgagag ccatgcatga ttagtggtta
2820ccctattgaa taggtcagaa gtagaatctt ttcatcatca cagaaagttc
tattggacag 2880tgctcttcta gatcatcata agactacaga gcacttttca
aagctcatgc atgttcatca 2940tgttagtgtc gtattt 29564302PRTHomo sapiens
4Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu1 5
10 15Ala Ala Pro Gln Glu Ala Leu Pro Asp Glu Thr Glu Val Val Glu
Glu 20 25 30Thr Val Ala Glu Val Thr Glu Val Ser Val Gly Ala Asn Pro
Val Gln 35 40 45Val Glu Val Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr
Glu Glu Glu 50 55 60Val Val Ala Glu Asn Pro Cys Gln Asn His His Cys
Lys His Gly Lys65 70 75 80Val Cys Glu Leu Asp Glu Asn Asn Thr Pro
Met Cys Val Cys Gln Asp 85 90 95Pro Thr Ser Cys Pro Ala Pro Ile Gly
Glu Phe Glu Lys Val Cys Ser 100 105 110Asn Asp Asn Lys Thr Phe Asp
Ser Ser Cys His Phe Phe Ala Thr Lys 115 120 125Cys Thr Leu Glu Gly
Thr Lys Lys Gly His Lys Leu His Leu Asp Tyr 130 135 140Ile Gly Pro
Cys Lys Tyr Ile Pro Pro Cys Leu Asp Ser Glu Leu Thr145 150 155
160Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Val Leu Val Thr
165 170 175Leu Tyr Glu Arg Asp Glu Asp Asn Asn Leu Leu Thr Glu Lys
Gln Lys 180 185 190Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg
Leu Glu Ala Gly 195 200 205Asp His Pro Val Glu Leu Leu Ala Arg Asp
Phe Glu Lys Asn Tyr Asn 210 215 220Met Tyr Ile Phe Pro Val His Trp
Gln Phe Gly Gln Leu Asp Gln His225 230 235 240Pro Ile Asp Gly Tyr
Leu Ser His Thr Glu Leu Ala Pro Leu Arg Ala 245 250 255Pro Leu Ile
Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu Thr Cys 260 265 270Asp
Leu Asp Asn Asp Lys Tyr Ile Ala Leu Asp Glu Trp Ala Gly Cys 275 280
285Phe Gly Ile Lys Gln Lys Asp Ile Asp Lys Asp Leu Val Ile 290 295
3005909DNAHomo sapiens 5atgagggcct ggatcttctt tctcctttgc ctggccggga
gggccttggc agcccctcaa 60gaagccctgc ctgatgagac agaggtggtg gaagaaactg
tggcagaggt gactgaggta 120tctgtgggag ctaatcctgt ccaggtggaa
gtaggagaat ttgatgatgg tgcagaggaa 180accgaagagg aggtggtggc
ggaaaatccc tgccagaacc accactgcaa acacggcaag 240gtgtgcgagc
tggatgagaa caacaccccc atgtgcgtgt gccaggaccc caccagctgc
300ccagccccca ttggcgagtt tgagaaggtg tgcagcaatg acaacaagac
cttcgactct 360tcctgccact tctttgccac aaagtgcacc ctggagggca
ccaagaaggg ccacaagctc 420cacctggact acatcgggcc ttgcaaatac
atcccccctt gcctggactc tgagctgacc 480gaattccccc tgcgcatgcg
ggactggctc aagaacgtcc tggtcaccct gtatgagagg 540gatgaggaca
acaaccttct gactgagaag cagaagctgc gggtgaagaa gatccatgag
600aatgagaagc gcctggaggc aggagaccac cccgtggagc tgctggcccg
ggacttcgag 660aagaactata acatgtacat cttccctgta cactggcagt
tcggccagct ggaccagcac 720cccattgacg ggtacctctc ccacaccgag
ctggctccac tgcgtgctcc cctcatcccc 780atggagcatt gcaccacccg
ctttttcgag acctgtgacc tggacaatga caagtacatc 840gccctggatg
agtgggccgg ctgcttcggc atcaagcaga aggatatcga caaggatctt 900gtgatctaa
9096858DNAHomo sapiens 6gcccctcaag aagccctgcc tgatgagaca gaggtggtgg
aagaaactgt ggcagaggtg 60actgaggtat ctgtgggagc taatcctgtc caggtggaag
taggagaatt tgatgatggt 120gcagaggaaa ccgaagagga ggtggtggcg
gaaaatccct gccagaacca ccactgcaaa 180cacggcaagg tgtgcgagct
ggatgagaac aacaccccca tgtgcgtgtg ccaggacccc 240accagctgcc
cagcccccat tggcgagttt gagaaggtgt gcagcaatga caacaagacc
300ttcgactctt cctgccactt ctttgccaca aagtgcaccc tggagggcac
caagaagggc 360cacaagctcc acctggacta catcgggcct tgcaaataca
tccccccttg cctggactct 420gagctgaccg aattccccct gcgcatgcgg
gactggctca agaacgtcct ggtcaccctg 480tatgagaggg atgaggacaa
caaccttctg actgagaagc agaagctgcg ggtgaagaag 540atccatgaga
atgagaagcg cctggaggca ggagaccacc ccgtggagct gctggcccgg
600gacttcgaga agaactataa catgtacatc ttccctgtac actggcagtt
cggccagctg 660gaccagcacc ccattgacgg gtacctctcc cacaccgagc
tggctccact gcgtgctccc 720ctcatcccca tggagcattg caccacccgc
tttttcgaga cctgtgacct ggacaatgac 780aagtacatcg ccctggatga
gtgggccggc tgcttcggca tcaagcagaa ggatatcgac 840aaggatcttg tgatctaa
858722DNAHomo sapiens 7agggccttgg cagcccctca ag 22823DNAHomo
sapiens 8agggccttgg cagcccctca gca 23923DNAHomo sapiens 9gaaactgtgg
cagaggtgac tga 231025DNAHomo sapiens 10gttgtcattg ctgcacacct tctca
25112956DNAHomo sapiens 11ctccacattc ccgcggtcct tcagactgcc
cggagagcgc gctctgcctg ccgcctgcct 60gcctgccact gagggttccc agcaccatga
gggcctggat cttctttctc ctttgcctgg 120ccgggagggc cttggcagcc
cctcaagaag ccctgcctga tgagacagag gtggtggaag 180aaactgtggc
agaggtgact gaggtatctg tgggagctaa tcctgtccag gtggaagtag
240gagaatttga tgatggtgca gaggaaaccg aagaggaggt ggtggcggaa
aatccctgcc 300agaaccacca ctgcaaacac ggcaaggtgt gcgagctgga
tgagaacaac acccccatgt 360gcgtgtgcca ggaccccacc agctgcccag
cccccattgg cgagtttgag aaggtgtgca 420gcaatgacaa caagaccttc
gactcttcct gccacttctt tgccacaaag tgcaccctgg 480agggcaccaa
gaagggccac aagctccacc tggactacat cgggccttgc aaatacatcc
540ccccttgcct ggactctgag ctgaccgaat tccccctgcg catgcgggac
tggctcaaga 600acgtcctggt caccctgtat gagagggatg aggacaacaa
ccttctgact gagaagcaga 660agctgcgggt gaagaagatc catgagaatg
agaagcgcct ggaggcagga gaccaccccg 720tggagctgct ggcccgggac
ttcgagaaga actataacat gtacatcttc cctgtacact 780ggcagttcgg
ccagctggac cagcacccca ttgacgggta cctctcccac accgagctgg
840ctccactgcg tgctcccctc atccccatgg agcattgcac cacccgcttt
ttcgagacct 900gtgacctgga caatgacaag tacatcgccc tggatgagtg
ggccggctgc ttcggcatca 960agcagaagga tatcgacaag gatcttgtga
tctaaatcca ctccttccac agtaccggat 1020tctctcttta accctcccct
tcgtgtttcc cccaatgttt aaaatgtttg gatggtttgt 1080tgttctgcct
ggagacaagg tgctaacata gatttaagtg aatacattaa cggtgctaaa
1140aatgaaaatt ctaacccaag acatgacatt cttagctgta acttaactat
taaggccttt 1200tccacacgca ttaatagtcc catttttctc ttgccatttg
tagctttgcc cattgtctta 1260ttggcacatg ggtggacacg gatctgctgg
gctctgcctt aaacacacat tgcagcttca 1320acttttctct ttagtgttct
gtttgaaact aatacttacc gagtcagact ttgtgttcat 1380ttcatttcag
ggtcttggct gcctgtgggc ttccccaggt ggcctggagg tgggcaaagg
1440gaagtaacag acacacgatg ttgtcaagga tggttttggg actagaggct
cagtggtggg 1500agagatccct gcagaaccca ccaaccagaa cgtggtttgc
ctgaggctgt aactgagaga 1560aagattctgg ggctgtgtta tgaaaatata
gacattctca cataagccca gttcatcacc 1620atttcctcct ttacctttca
gtgcagtttc ttttcacatt aggctgttgg ttcaaacttt 1680tgggagcacg
gactgtcagt tctctgggaa gtggtcagcg catcctgcag ggcttctcct
1740cctctgtctt ttggagaacc agggctcttc tcaggggctc tagggactgc
caggctgttt 1800cagccaggaa ggccaaaatc aagagtgaga tgtagaaagt
tgtaaaatag aaaaagtgga 1860gttggtgaat cggttgttct ttcctcacat
ttggatgatt gtcataaggt ttttagcatg 1920ttcctccttt tcttcaccct
cccctttttt cttctattaa tcaagagaaa cttcaaagtt 1980aatgggatgg
tcggatctca caggctgaga actcgttcac ctccaagcat ttcatgaaaa
2040agctgcttct tattaatcat acaaactctc accatgatgt gaagagtttc
acaaatcctt 2100caaaataaaa agtaatgact tagaaactgc cttcctgggt
gatttgcatg tgtcttagtc 2160ttagtcacct tattatcctg acacaaaaac
acatgagcat acatgtctac acatgactac 2220acaaatgcaa acctttgcaa
acacattatg cttttgcaca cacacacctg tacacacaca 2280ccggcatgtt
tatacacagg gagtgtatgg ttcctgtaag cactaagtta gctgttttca
2340tttaatgacc tgtggtttaa cccttttgat cactaccacc attatcagca
ccagactgag 2400cagctatatc cttttattaa tcatggtcat tcattcattc
attcattcac aaaatattta 2460tgatgtattt actctgcacc aggtcccatg
ccaagcactg gggacacagt tatggcaaag 2520tagacaaagc atttgttcat
ttggagctta gagtccagga ggaatacatt agataatgac 2580acaatcaaat
ataaattgca agatgtcaca ggtgtgatga agggagagta ggagagacca
2640tgagtatgtg taacaggagg acacagcatt attctagtgc tgtactgttc
cgtacggcag 2700ccactaccca catgtaactt tttaagattt aaatttaaat
tagttaacat tcaaaacgca 2760gctccccaat cacactagca acatttcaag
tgcttgagag ccatgcatga ttagtggtta 2820ccctattgaa taggtcagaa
gtagaatctt ttcatcatca cagaaagttc tattggacag 2880tgctcttcta
gatcatcata agactacaga gcacttttca aagctcatgc atgttcatca
2940tgttagtgtc gtattt 2956122968DNAHomo sapiens 12ctccacattc
ccgcggtcct tcagactgcc cggagagcgc gctctgcctg ccgcctgcct 60gcctgccact
gagggttccc agcaccatga gggcctggat cttctttctc ctttgcctgg
120ccgggagggc cttggcagcc cctcagcaag aagccctgcc tgatgagaca
gaggtggtgg 180aagaaactgt ggcagaggtg actgaggtat ctgtgggagc
taatcctgtc caggtggaag 240taggagaatt tgatgatggt gcagaggaaa
ccgaagagga ggtggtggcg gaaaatccct 300gccagaacca ccactgcaaa
cacggcaagg tgtgcgagct ggatgagaac aacaccccca 360tgtgcgtgtg
ccaggacccc accagctgcc cagcccccat tggcgagttt gagaaggtgt
420gcagcaatga caacaagacc ttcgactctt cctgccactt ctttgccaca
aagtgcaccc 480tggagggcac caagaagggc cacaagctcc acctggacta
catcgggcct tgcaaataca 540tccccccttg cctggactct gagctgaccg
aattccccct gcgcatgcgg gactggctca 600agaacgtcct ggtcaccctg
tatgagaggg atgaggacaa caaccttctg actgagaagc 660agaagctgcg
ggtgaagaag atccatgaga atgagaagcg cctggaggca ggagaccacc
720ccgtggagct gctggcccgg gacttcgaga agaactataa catgtacatc
ttccctgtac 780actggcagtt cggccagctg gaccagcacc ccattgacgg
gtacctctcc cacaccgagc 840tggctccact gcgtgctccc ctcatcccca
tggagcattg caccacccgc tttttcgaga 900cctgtgacct ggacaatgac
aagtacatcg ccctggatga gtgggccggc tgcttcggca 960tcaagcagaa
ggatatcgac aaggatcttg tgatctaaat ccactccttc cacagtaccg
1020gattctctct ttaaccctcc ccttcgtgtt tcccccaatg tttaaaatgt
ttggatggtt 1080tgttgttctg cctggagaca aggtgctaac atagatttaa
gtgaatacat taacggtgct 1140aaaaatgaaa attctaaccc aagacatgac
attcttagct gtaacttaac tattaaggcc 1200ttttccacac gcattaatag
tcccattttt ctcttgccat ttgtagcttt gcccattgtc 1260ttattggcac
atgggtggac acggatctgc tgggctctgc cttaaacaca cattgcagct
1320tcaacttttc
tctttagtgt tctgtttgaa actaatactt accgagtcag actttgtgtt
1380catttcattt cagggtcttg gctgcctgtg ggcttcccca ggtggcctgg
aggtgggcaa 1440agggaagtaa cagacacacg atgttgtcaa ggatggtttt
gggactagag gctcagtggt 1500gggagagatc cctgcagaac ccaccaacca
gaacgtggtt tgcctgaggc tgtaactgag 1560agaaagattc tggggctgtg
ttatgaaaat atagacattc tcacataagc ccagttcatc 1620accatttcct
cctttacctt tcagtgcagt ttcttttcac attaggctgt tggttcaaac
1680ttttgggagc acggactgtc agttctctgg gaagtggtca gcgcatcctg
cagggcttct 1740cctcctctgt cttttggaga accagggctc ttctcagggg
ctctagggac tgccaggctg 1800tttcagccag gaaggccaaa atcaagagtg
agatgtagaa agttgtaaaa tagaaaaagt 1860ggagttggtg aatcggttgt
tctttcctca catttggatg attgtcataa ggtttttagc 1920atgttcctcc
ttttcttcac cctccccttt tttcttctat taatcaagag aaacttcaaa
1980gttaatggga tggtcggatc tcacaggctg agaactcgtt cacctccaag
catttcatga 2040aaaagctgct tcttattaat catacaaact ctcaccatga
tgtgaagagt ttcacaaatc 2100cttcaaaata aaaagtaatg acttagaaac
tgccttcctg ggtgatttgc atgtgtctta 2160gtcttagtca ccttattatc
ctgacacaaa aacacatgag catacatgtc tacacatgac 2220tacacaaatg
caaacctttg caaacacatt atgcttttgc acacacacac ctgtacacac
2280acaccggcat gtttatacac agggagtgta tggttcctgt aagcactaag
ttagctgttt 2340tcatttaatg acctgtggtt taaccctttt gatcactacc
accattatca gcaccagact 2400gagcagctat atccttttat taatcatggt
cattcattca ttcattcatt cacaaaatat 2460ttatgatgta tttactctgc
accaggtccc atgccaagca ctggggacac agttatggca 2520aagtagacaa
agcatttgtt catttggagc ttagagtcca ggaggaatac attagataat
2580gacacaatca aatataaatt gcaagatgtc acaggtgtga tgaagggaga
gtaggagaga 2640ccatgagtat gtgtaacagg aggacacagc attattctag
tgctgtactg ttccgtacgg 2700cagccactac ccacatgtaa ctttttaaga
tttaaattta aattagttaa cattcaaaac 2760gcagctcccc aatcacacta
gcaacatttc aagtgcttga gagccatgca tgattagtgg 2820ttaccctatt
gaataggtca gaagtagaat cttttcatca tcacagaaag ttctattgga
2880cagtgctctt ctagatcatc ataagactac agagcacttt tcaaagctca
tgcatgttca 2940tcatgttagt gtcgtatttg tcgtattt 296813113DNAHomo
sapiens 13ctccacattc ccgcggtcct tcagactgcc cggagagcgc gctctgcctg
ccgcctgcct 60gcctgccact gagggttccc agcaccatga gggcctggat cttctttctc
ctt 11314113DNAHomo sapiens 14ctccacattc ccgcggtcct tcagactgcc
cggagagcgc gctctgcctg ccgcctgcct 60gcctgccact gagggttccc agcaccatga
gggcctggat cttctttctc ctt 11315871DNAHomo sapiens 15ctccacattc
ccgcggtcct tcagactgcc cggagagcgc gctctgcctg ctccacattc 60ccgcggtcct
tcagactgcc cggagagcgc gctctgcctg cttctttctc ctttgcctgg
120ccgggagggc cttggcagcc cctcaagaag ccctgcctga tgagacagag
gtggtggaag 180aaactgtggc agaggtgact gaggtatctg tgggagctaa
tcctgtccag gtggaagtag 240gagaatttga tgatggtgca gaggaaaccg
aagaggaggt ggtggcggaa aatccctgcc 300agaaccacca ctgcaaacac
ggcaaggtgt gcgagctgga tgagaacaac acccccatgt 360gcgtgtgcca
ggaccccacc agctgcccag cccccattgg cgagtttgag aaggtgtgca
420gcaatgacaa caagaccttc gactcttcct gccacttctt tgccacaaag
tgcaccctgg 480agggcaccaa gaagggccac aagctccacc tggactacat
cgggccttgc aaatacatcc 540ccccttgcct ggactctgag ctgaccgaat
tccccctgcg catgcgggac tggctcaaga 600acgtcctggt caccctgtat
gagagggatg aggacaacaa ccttctgact gagaagcaga 660agctgcgggt
gaagaagatc catgagaatg agaagcgcct ggaggcagag accaccccgt
720ggagctgctg gccccggact tcgagaagac tataacatgt acatcctccc
tgtacactgg 780cagttcggcc agctggacca gcaccccatt gacgggtacc
tctcccacac cgagctggct 840ccatgcgtgc tcccctcatc ccatggagca t
87116757DNAHomo sapiensmisc_feature(721)..(722)n is a, c, g, or t
16ctccacattc ccgcggtcct tcagactgcc cggagagcgc gctctgcctg ccgcctgcct
60gcctgccact gagggttccc agcaccatga gggcctggat cttctttctc ctttgcctgg
120ccgggagggc cttggcagcc cctcaagaag ccctgcctga tgagacagag
gtggtggaag 180aaactgtggc agaggtgtga tgatggtgca gaggaaaccg
aagaggaggt ggtggcggaa 240aatccctaac acccccatgt gcgtgtgcca
ggaccccacc agctgcccag cccccattgg 300cgagtttgag aaggtgtgca
gcaatgacaa caagaccttc gactcttcct gccacttctt 360tgccacaaag
tgcaccctgg agggcaccaa gaagggccac aagctccacc tggactacat
420cgggccttgc aaatacatcc ccccttgaga acgtcctggt caccctgtat
gagagggatg 480aggacaacaa ccttctgact gagaagcaga agctgcgggt
gaagaagatc catgagaatg 540agaagcgcct ggaggcagga gaccaccccg
tggagctgct ggcccgggac ttcgagaaga 600actataacat gtacatcttc
cctgtacact ggcagttcgg ccagctggac cagcacccca 660ttgacgggta
cctctcccac accgagctgg ctccactgcg tgctcccctc atccccatgg
720nncattgcac cacccgcttt ttcgagacct gtgacct 75717975DNAHomo
sapiensmisc_feature(34)..(34)n is a, c, g, or t 17tgagggttcc
cagcaccatg agggcctgga tctntttctc ctttgcctgg ncgggagggc 60cttggcagcc
cctcaagaag ccctgcctga tgagacagag gtggtggaag aaactgtggc
120agaggtgact gaggtatctg tgggagctaa tcctgnncag gtggaagtag
gagaatttga 180tgatggtgca gaggaaaccg aagaggaggt ggtggcggaa
aatccctgcc agaaccacca 240ctgcaaacac ggcaaggtgt gcgagctgga
tgagaacaac acccccatgt gcgtgtgcca 300ggaccccacc agctgcccag
cccccattgg cgagtttgag aaggtgtgca gcaatgacaa 360caagaccttc
gactcttcct gccacttctt tgccacaaag tgcaccctgg agggcaccaa
420gaagggccac aagctccacc tggactacat cgggccttgc aaatacatcc
ccccttgcct 480ggactttgag ctgaccgaat tccccctgcg catgcgggac
tggctcaaga acgtcctggt 540caccctgtat gagagggatg aggacaacaa
ccttttgact gagaagcaga agctgcgggt 600gaagaagatc catgagaatg
agaagcgcct ggaggcagga gaccaccccg tggagctgct 660ggcccgggac
ttcgagaaga actataacat gtacattttc cctgtacact ggcagttcgg
720ccagctggac cagcacccca ttgacgggta cctctcccac accgagctgg
ctccactgcg 780tgctcccctc atccccatgg agcattgcac cacccgcttt
ttcgagacct gtgacctgga 840caatgacaag tacatcgccc tggatgagtg
ggccggctgc ttcggcatca agcagaagga 900tatcgacaag gatcttgtga
tttaaatcca ctccttccac agtaccggat tctctcttta 960accctcccct tcgtg
97518938DNAHomo sapiensmisc_feature(24)..(24)n is a, c, g, or t
18tcccagcacc atgaggcctg gatnttcttt ntcctttgcc tggccgggag gncttggcag
60ccctcaagaa gccctgcctg atgagacaga ggtggtggaa gaaactgtgg cagaggtgac
120tgaggtatct gtgggagcta atcctgncag gtggaagtag gagaatttga
tgatggtgca 180gaggaaacga agaggaggtg gtggcggaaa atccctgcca
gaaccaccac tgcaaacacg 240gcaaggtgtg cgagctggat gagaacaaca
cccccatgtg cgtgtgccag gaccccacca 300gctgcccagc ccccattggc
gagtttgaga aggtgtgcag caatgacaac aagaccttcg 360actcttcctg
ccacttcttt gccacaaagt gcaccctgga gggcaccaag aagggccaca
420agctccacct ggactacatc gggccttgca aatacatccc cccttgcctg
gactttgagc 480tgaccgaatt ccccctgcgc atgcgggact ggctcaagaa
cgtcctggtc accctgtatg 540agagggatga ggacaacaac cttttgactg
agaagcagaa gctgcgggtg aagaagatcc 600atgagaatga gaagcgcctg
gaggcaggag accaccccgt ggagctgctg gcccgggact 660tcgagaagaa
ctataacatg tacattttcc ctgtacactg gcagttcggc cagctggacc
720agcaccccat tgacgggtac ctttcccaca ccgagctggc tccactgcgt
gctcccctca 780tccccatgga gcattgcacc acccgctttt tcgagacctg
tgacctggac aatgacaagt 840acatcgccct ggatgagtgg gccggctgct
tcggcatcaa gcagaaggat atcgacaagg 900atcttgtgat ttaaatccac
tccttccaca gtaccgga 93819852DNAHomo
sapiensmisc_feature(803)..(803)n is a, c, g, or t 19aaccttctga
ctgagaagca gaagctgcgg gtgaagaaga tccatgagaa tgagaagcgc 60ctggaggcag
gagaccaccc cgtggagctg ctggcccggg acttcgagaa gaactataac
120atgtacatct tccctgtaca ctggcagttc ggccagctgg accagcaccc
cattgacggg 180tacctctccc acaccgagct ggctccactg cgtgctcccc
tcatccccat ggagcattgc 240accacccgct ttttcgagac ctgtgacctg
gacaatgaca agtacatcgc cctggatgag 300tgggccggct gcttcggcat
caagcagaag gatatcgaca aggatcttgt gatctaaatc 360cactccttcc
acagtaccgg attctctctt taaccctccc cttcgtgttt cccccaatgt
420ttaaaatgtt tggatggttt gttgttctgc ctggagacaa ggtgctaaca
tagatttaag 480tgaatacatt aacggtgcta aaaatgaaaa ttctaaccca
agacatgaca ttcttagctg 540taacttaact attaaggcct tttccacacg
cattaatagt cccatttttc tcttgccatt 600tgtagctttg cccattgtct
tattggcaca tgggtggaca cggatctgct gggctctgcc 660ttaaacacac
attgcagctt caacttttct ctttagtgtt ctgtttgaaa ctaatactta
720ccgagtcaga ctttgtgttc atttcatttc agggtcttgg ctgcctgtgg
gcttccccag 780gtggcctgga ggtgggcaaa ggnaagtaac agacacacga
tgttgtcaan gatggtttgg 840gactagaggc tc 85220944DNAHomo
sapiensmisc_feature(833)..(833)n is a, c, g, or t 20gcgggtgaag
aagatccatg agaatgagaa gcgcctggag gcaggagacc accccgtgga 60gctgctggcc
cgggacttcg agaagaacta taacatgtac atcttccctg tacactggca
120gttcggccag ctggaccagc accccattga cgggtacctc tcccacaccg
agctggctcc 180actgcgtgct cccctcatcc ccatggagca ttgcaccacc
cgctttttcg agacctgtga 240cctggacaat gacaagtaca tcgccctgga
tgagtgggcc ggctgcttcg gcatcaagca 300gaaggatatc gacaaggatc
ttgtgatcta aatccactcc ttccacagta ccggattctc 360tctttaaccc
tccccttcgt gtttccccca atgtttaaaa tgtttggatg gtttgttgtt
420ctgcctggag acaaggtgct aacatagatt taagtgaata cattaacggt
gctaaaaatg 480aaaattctaa cccaagacat gacattctta gctgtaactt
aactattaag gccttttcca 540cacgcattaa tagtcccatt tttctcttgc
catttgtagc tttgcccatt gtcttattgg 600cacatgggtg gacacggatc
tgctgggctc tgccttaaac acacattgca gcttcaactt 660ttctctttag
tgttctgttt gaaactaata cttaccgagt cagactttgt gttcatttca
720tttcagggtc ttggctgcct gtgggcttcc ccaggtggcc tggaggtggg
caaagggaag 780taacagacac acgatgttgt cagggatggt tttgggacta
gaggctcagt ggngggagag 840atcnctgcag aanncnncaa ccagaacgtg
gtttgcctga gntgtaactg agagaanatt 900tctggggctg tgtatgaaaa
tatagacntt ctcacataag ccca 94421985DNAHomo
sapiensmisc_feature(8)..(8)n is a, c, g, or t 21tggcacangg
gtggacacgg atctgctggg ntctgcctta aacncacatt gcagntcaac 60ttttctcttt
agtgttctgt ttgaaactaa tacttaccga gtcagacttt gtgttcattt
120catttcaggg tcttggctgc ctgtgggctt ccccaggtgg cctggaggtg
ggcaaaggga 180agtaacagac acacgatgtt gtcaaggatg gttttgggac
tagaggctca gtggtgggag 240agatccctgc agaacccacc caccagaacg
tggtttgcct gaggctgtaa ctgagagaaa 300gattctgggg ctgtgttatg
aaaatataga cattctcaca taagcccagt tcatcaccat 360ttcctccttt
acctttcagt gcagtttctt ttcacattag gctgttggtt caaacttttg
420ggagcacgga ctgtcagttc tctgggaagt ggtcagcgca tcctgcaggg
cttctcctcc 480tctgtctttt ggagaaccag ggctcttctc aggggctcta
gggactgcca ggctgtttca 540gccaggaagg ccaaaatcaa gagtgagatg
tagaaagttg taaaatagaa aaagtggagt 600tggtgaatcg gttgttcttt
cctcacattt ggatgattgt cataaggttt ttagcatgtt 660cctccttttc
ttcaccctcc ccttttttct tctattaatc aagagaaact tcaaagttaa
720tgggatggtc ggatctcaca ggctgagaac tcgttcacct ccaagcattt
catgaaaaag 780ctgcttctta ttaatcatac aaactctcac catgatgtga
agagtttcac aaatccttca 840aaataaaaag taatgactta gaaactgcct
tcctgggtga tttgcatgtg tcttagtctt 900agtcacctta ttatcctgac
acaaaaacac atgagcatac atgtctacac atgactacac 960aaatgcaaac
ctttgcaaac acatt 98522946DNAHomo sapiensmisc_feature(20)..(21)n is
a, c, g, or t 22gtttgaaact aatacttacn nagtcagact ttgtgttcat
ttcatttcag ggtctgggct 60gcctgtgggc ttccccaggt ggcctggagg tgggcaaagg
gaagtaacag acacacgatg 120ttgtcaagga tggttttggg actagaggct
cagtggtggg agagatccct gcagaaccca 180ccaaccagaa cgtgtttgcc
tgaggctgta actgagagaa agattctggg gctgtgttat 240gaaaatatag
acattctcac ataagcccag ttcatcacca tttcctcctt tacctttcag
300tgcagtttct tttcacatta ggctgttggt tcaaactttt gggagcacgg
actgtcagtt 360ctctgggaag tggtcagcgc atcctgcagg gcttctcctc
ctctgtcttt tggagaacca 420gggctcttct caggggctct agggactgcc
aggctgtttc agccaggaag gccaaaatca 480agagtgagat gtagaaagtt
gtaaaataga aaaagtggag ttggtgaatc ggttgttctt 540tcctcacatt
tggatgattg tcataaggtt tttagcatgt tcctcctttt cttcaccctc
600cccttttttc ttctattaat caagagaaac ttcaaagtta atgggatggt
cggatctcac 660aggctgagaa ctcgttcacc tccaagcatt tcatgaaaaa
gctgcttctt attaatcata 720caaactctca ccatgatgtg aagagtttca
caaatccttc aaaataaaaa gtaatgactt 780agaaactgcc ttcctgggtg
atttgcatgt gtcttagtct tagtcacctt attatcctga 840cacaaaaaca
catgagcata catgtctaca catgactaca caaatgcaaa cctttgcaaa
900cacattatgc ttttgcacac acacacctgt acacacacac cggcat
94623915DNAHomo sapiensmisc_feature(896)..(896)n is a, c, g, or t
23gctgcttctt attaatcata caaactctca ccatgatgtg aagagtttca caaatccttc
60aaaataaaaa gtaatgactt agaaactgcc ttcctgggtg atttgcatgt gtcttagtct
120tagtcacctt attatcctga cacaaaaaca catgagcata catgtctaca
catgactaca 180caaatgcaaa cctttgcaaa cacattatgc ttttgcacac
acacacctgt acacacacac 240cggcatgttt atacacaggg agtgtatggt
tcctgtaagc actaagttag ctgttttcat 300ttaatgacct gtggtttaac
ccttttgatc actaccacca ttatcagcac cagactgagc 360agctatatcc
ttttattaat catggtcatt cattcattca ttcattcaca aaatatttat
420gatgtattta ctctgcacca ggtcccatgc caagcactgg ggacacagtt
atggcaaagt 480agacaaagca tttgttcatt tggagcttag agtccaggag
gaatacatta gataatgaca 540caatcaaata taaattgcaa gatgtcacag
gtgtgatgaa gggagagtag gagagaccat 600gagtatgtgt aacaggagga
cacagcatta ttctagtgct gtactgttcc gtacggcagc 660cactacccac
atgtaacttt ttaagattta aatttaaatt agttaacatt caaaacgcag
720ctccccaatc acactagcaa catttcaagt gcttgagagc catgcatgat
tagtggttac 780cctattgaat aggtcagaag tagaatcttt tcatcatcac
agaaagttct attggacagt 840gctcttctag atcatcataa gactacagag
cacttttcaa agctcatgca tgttcntcat 900gttagtgtcg tattt
91524666DNAHomo sapiensmisc_feature(86)..(86)n is a, c, g, or t
24gtttataccc agggagggta ggtttcttgt aagcaataag ttagctgttt tcatttaagg
60acctgtggtt tacccttttg atcatntccc ccaatatcag caccagantg agcagctata
120tcttttatta tcatggtcat tcattcattc attcattcac aaaatattta
tgatgtattt 180actntgcacc aggtcccatg ccaagcactg gggacacagt
tatggcaaag tagacaaagc 240atttgttcat ttggagctta gagtccagga
ggaatacatt agataatgac acaatcaaat 300ataaattgca agatgtcaca
ggtgtgatga agggagagta ggagagacca tgagtatgtg 360taacaggagg
acacagcatt attctagtgc tgtactgttc cgtacggcag ccactaccca
420catgtaactt tttaagattt aaatttaaat tagttaacat tcaaaacgca
gctccccaat 480cacactagca acatttcaag tgcttgagag ccatgcatga
ttagtggtta ccctattgaa 540taggtcagaa gtagaatctt ttcatcatca
cagaaagttc tattggacag tgctcttcta 600gatcatcata agactacaga
gcacttttca aagctcatgc atgttcatca tgttagtgtc 660gtattt 666
* * * * *