U.S. patent application number 11/669725 was filed with the patent office on 2008-02-28 for mt-sp1 polypeptides.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Charles S. Craik, Marc Shuman, Toshihiko Takeuchi.
Application Number | 20080051559 11/669725 |
Document ID | / |
Family ID | 23624393 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051559 |
Kind Code |
A1 |
Craik; Charles S. ; et
al. |
February 28, 2008 |
MT-SP1 polypeptides
Abstract
This invention provides a novel membrane-type serine protease
(designated MT-SP1) elevated expression of which is associated with
cancer. In one embodiment, this invention provides a method
obtaining a prognosis or of detecting or staging a cancer in an
organism. The method involves providing a biological sample from
the organism and detecting the level of a membrane type serine
protease 1 (MT-SP1) in the sample, where an elevated level of the
membrane-type serine protease, as compared to the level of the
protease in a biological sample from a normal healthy organism
indicates the presence or stage of the cancer.
Inventors: |
Craik; Charles S.; (San
Francisco, CA) ; Takeuchi; Toshihiko; (San Francisco,
CA) ; Shuman; Marc; (San Francisco, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
23624393 |
Appl. No.: |
11/669725 |
Filed: |
January 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11253869 |
Oct 18, 2005 |
7227009 |
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11669725 |
Jan 31, 2007 |
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09410362 |
Sep 30, 1999 |
7030231 |
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11669725 |
Jan 31, 2007 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C12N 9/6408 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0001] This work was supported, in part, by National Institutes of
Health Grants Numbers CA72006 and CA71097. The Government of the
United States of America may have some rights in this invention.
Claims
1-80. (canceled)
81. An isolated polypeptide consisting of a serine proteinase
domain that is at least 95% identical to the amino acid sequence
from residue 615 to 855 shown in SEQ ID NO. 2 and optionally a
fusion protein thereto, wherein said domain exhibits serine
proteinase activity.
82. The isolated polypeptide of claim 81, wherein the MT-SP1 serine
protease domain exhibits specific protease activity towards a
target substrate.
83. The isolated polypeptide of claim 81, wherein said MT-SP1
serine protease domain has the sequence of amino acids 615 through
855 of SEQ ID NO:2.
84. The polypeptide of claim 81, wherein the target substrate has
the formula of Suc-AAPX-pNA, wherein: Suc is N-succinyl, A is
alanyl, P is prolyl pNA is paranitroanaline acetate X is selected
from the group consisting of alanyl, aspartyl, glutamyl,
phenylalanyl, leucinyl, methionyl, and arginyl residue.
85. The polypeptide of claim 84 having a k.sub.cat of about
2.6.times.10.sup.2/second or greater in a protease reaction
utilizing Spectozyme tPA as the target substrate.
86. The polypeptide of claim 84 having the sequence of amino acids
615 through 855 of SEQ ID NO:2.
87. An isolated polypeptide comprising an isolated MT-SP1 serine
proteinase domain that is substantially free of MT-SP1 sequences
from domains other than the serine proteinase domain.
88. The isolated polypeptide of claim 87, wherein the isolated
MT-SP1 serine proteinase domain comprises C-terminal sequence of
MT-SP1 having a length less than about 250 amino acids.
89. The isolated polypeptide of claim 87 that exhibits serine
proteinase activity.
90. The isolated polypeptide of claim 87 that substantially lacks
serine proteinase activity.
91. The isolated polypeptide of claim 81, wherein the fusion
protein comprises an epitope tag.
92. The isolated polypeptide of claim 81 that is labeled with a
detectable label.
93. The isolated polypeptide of claim 87 that is labeled with a
detectable label.
94. The isolated polypeptide of claim 81 that is bound to an
anti-MT-SP1 antibody.
95. The isolated polypeptide of claim 87 that is bound to an
anti-MT-SP1 antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] [Not Applicable]
FIELD OF THE INVENTION
[0003] This invention relates to the field of serine proteases and
associated biology. In particular, this invention relates to the
discovery of a new membrane-type serine protease believed to be
associated with the etiology of cancer and associated
pathologies.
BACKGROUND OF THE INVENTION
[0004] The serine proteases (SP) are a large family of proteolytic
enzymes that include the digestive enzymes, trypsin and
chymotrypsin, components of the complement cascade and of the
blood-clotting cascade, and enzymes that control the degradation
and turnover of macromolecules of the extracellular matrix. Serine
proteases are so named because of the presence of a serine residue
in the active catalytic site for protein cleavage. Serine proteases
have a wide range of substrate specificities and can be subdivided
into subfamilies on the basis of these specificities. The main
sub-families are trypases (cleavage after arginine or lysine),
aspases (cleavage after aspartate), chymases (cleavage after
phenylalanine or leucine), metases (cleavage after methionine), and
serases (cleavage after serine).
[0005] Most proteases are secretory proteins which contain
N-terminal signal peptides that serve to export the immature
protein across the endoplasmic reticulum and are then cleaved (von
Heijne (1986) Nuc. Acid. Res. 14: 5683-5690). Differences in these
signal sequences provide one means of distinguishing individual
serine proteases. Some serine proteases, particularly the digestive
enzymes, exist as inactive precursors or preproenzymes, and contain
a leader or activation peptide sequence 3' of the signal peptide.
Typically, this activation peptide may be 2-12 amino acids in
length, and it extends from the cleavage site of the signal peptide
to the N-terminal IIGG sequence of the active, mature protein.
Cleavage of this sequence activates the enzyme. This sequence
varies in different serine proteases according to the biochemical
pathway and/or its substrate (Zunino et al. (1988) Biochimica et.
Biophysica Acta 967: 331-340; Sayers, et al. (1992) J. Immunology
148: 292-300). Other features that distinguish various serine
proteases are the presence or absence of N-linked glycosylation
sites that provide membrane anchors, the number and distribution of
cysteine residues that determine the secondary structure of the
serine protease and the sequence of a substrate binding sites such
as S'. The S' substrate binding region is defined by residues
extending from approximately +17 to +29 relative to the N-terminal
I (+1). Differences in this region of the molecule are believed to
determine serine protease substrate specificities (Zunino et al,
supra).
[0006] Numerous disease states are caused by and can be
characterized by alterations in the activity of specific proteases
and their inhibitors. For example emphysema, arthritis, thrombosis,
cancer metastasis and some forms of hemophilia result from the lack
of regulation of serine protease activities (see, for example,
Textbook of Biochemistry with Clinical Correlations, John Wiley and
Sons, Inc. N.Y. (1993)). In case of viral infection, the presence
of viral proteases have been identified in infected cells. Such
viral proteases include, for example, HIV protease associated with
AIDS and NS3 protease associated with Hepatitis C. These viral
proteases play a critical role in the virus life cycle.
[0007] A series of serine proteases have been identified in murine
cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells. These
serine proteases are involved with CTL and NK cells in the
destruction of virally transformed cells and tumor cells and in
organ and tissue transplant rejection (Zunino et al. (1990) J.
Immunol. 144:2001-2009; Sayers et al. (1994) J. Immunol. 152:
2289-2297). Human homologs of most of these enzymes have been
identified (Trapaniet et al. (1988) Proc. Natl. Acad. Sci. 85:
6924-6928; Caputo et al. (1990) J. Immunol. 145: 737-744).
[0008] Proteases have also been implicated in cancer metastasis.
Increased synthesis of the protease urokinase has been correlated
with an increased ability to metastasize in many cancers. Urokinase
activates plasmin from plasminogen which is ubiquitously located in
the extracellular space and its activation can cause the
degradation of the proteins in the extracellular matrix through
which the metastasizing tumor cells invade. Plasmin can also
activate the collagenases thus promoting the degradation of the
collagen in the basement membrane surrounding the capillaries and
lymph system thereby allowing tumor cells to invade into the target
tissues (Dano, et al. (1985) Adv. Cancer. Res., 44: 139).
[0009] The discovery of a new serine protease precursor and the
polynucleotides encoding it satisfies a need in the art by
providing new prognostic and diagnostic methods and, therapeutic
compositions useful in the treatment or prevention of cancer.
SUMMARY OF THE INVENTION
[0010] This invention pertains to the discovery of a new serine
protease associated with cancer cells. In particular, nucleic acid
cDNAs derived from PC-3 mRNA were sequenced that encoded a novel
serine protease referred to herein as membrane-type serine protease
1 (MT-SP1). The MT-SP1 polypeptide encoded by the nucleic acid(s)
is localized in tumor tissues (e.g. prostatic cancers, gastric
cancers, breast cancers, etc.), and in preferred embodiments is
identified in blood vessels associated with tumors. Inhibition of
MT-SP1 inhibits cancer growth in relevant animal models. Without
being bound to a particular theory it is believed that MT-SP1 is
implicated in tumor proliferation and/or growth and/or tumor
angiogenesis. MT-SP1 is also demonstrated herein to be a good
diagnostic, and more preferably, a good prognostic for various
cancers. MT-SP1 can be used to detect the presence or absence of a
cancer, to determine the location and/or size and/or morphology of
a cancer, and to make a prediction regarding the severity and/or
outcome of a cancer or a particular therapeutic regimen.
[0011] In one embodiment, this invention provides nucleic acids
encoding MT-SP1 and/or probes suitable for amplification of MT-SP1
nucleic acids (e.g. from a PC-3 mRNA template). These nucleic acids
include, but are not limited to: (a) a nucleic acid comprising a
nucleic acid encoding a serine protease domain having the sequence
of SEQ ID NO: 2; (b) a nucleic acid comprising a nucleic acid
encoding a serine protease domain having the sequence of amino
acids 615 through 855 of SEQ ID NO: 2; (c) a nucleic acid that
specifically hybridizes to the nucleic acid of SEQ ID NO: 1 or a
fragment thereof under stringent conditions and is of sufficient
length that said nucleic acid can uniquely indicate the presence or
absence of a nucleic acid encoding a membrane-type serine protease
in a total genomic DNA pool, a total cDNA pool or a total mRNA pool
sample from a PC-3 cell; (d) a nucleic acid comprising a sequence
that has the same sequence as a nucleic acid amplified from a PC-3
cDNA template using PCR primers corresponding to nucleotides 37-54
of SEQ ID NO: 1 and 2604-2583 of the complement of SEQ ID NO: 1;
(e) a DNA encoding an mRNA that, when reverse transcribed, produces
the cDNA of SEQ ID NO: 1; (f) a DNA encoding an mRNA that, when
reverse transcribed, produces the cDNA encoding amino acids 615-855
of SEQ ID NO: 2; (g) a pair of primers that, when used in a nucleic
acid amplification reaction with PC-3 cDNA template specifically
amplifies a nucleic acid encoding the polypeptide of SEQ ID NO: 2;
(h) a pair of primers that, when used in a nucleic acid
amplification reaction with mRNA template from a PC-3 cell
specifically amplify a nucleic acid encoding the polypeptide having
the sequence of amino acids 615 through 855 of SEQ ID NO: 2; and
(i) a nucleic acid encoding a membrane-type serine protease,
wherein said nucleic acid encodes a consensus sequence shown in
FIG. 4 and does not encode TRYB_human, ENTK-Human, HEPS_human,
TRY2_Human, and CTRB_human. Preferred nucleic acids encode a
polypeptide having the sequence of amino acids 615 through 855 of
SEQ ID NO: 2, while other preferred nucleic acids encode a
polypeptide having the sequence of SEQ ID NO: 2. In one embodiment
the nucleic acid has the sequence of SEQ ID NO: 1. The nucleic
acid(s) are optionally present in an expression cassette and/or a
vector and are optionally labeled with a detectable label. Also
provided are host cells comprising such vectors and a process
producing a polypeptide comprising expressing from such host cells
a polypeptide encoded an MT-SP1 DNA. This invention also includes a
process for producing a cell that expresses an MT-SP1 polypeptide.
The process involves comprising transforming or transfecting the
cell with the vector encoding an MT-SP1 such that the cell
expresses the MT-SP1 polypeptide.
[0012] In another embodiment this invention provides isolated
MT-SP1 polypeptides (e.g. as encoded by the nucleic acids described
above). Preferred polypeptides comprise a protease domain of SEQ ID
NO: 2 or the polypeptide of SEQ ID NO: 2. Preferred polypeptides
also include, but are not limited to polypeptides that have serine
protease activity and that are specifically bound by an antibody
raised against the polypeptide of SEQ ID NO: 2 and/or polypeptides
having protease activity and having 95% or greater sequence
identity to a polypeptide having the sequence of SEQ ID NO: 2;
and/or having protease activity and having 95% or greater identity
to a polypeptide having the sequence of amino acids 615 through 855
of SEQ ID NO: 2.
[0013] Also provided are antibodies that specifically bind to the
MT-SP1 polypeptides of this invention (e.g. a polypeptide encoded
by SEQ ID NO: 2). The antibodies can be monoclonal, polyclonal,
antibody fragments or single-chain antibodies.
[0014] This invention also provides diagnostic assays for
cancer(s). Such assays involve providing a biological sample from
an organism; and detecting the level of a membrane type serine
protease 1 (MT-SP1) in the sample, where an elevated level of the
membrane-type serine protease, as compared to the level of the
protease in a biological sample from a normal healthy organism
indicates the presence of the cancer. The method can involve
determining the copy number of MT-SP1 genes in the cells of the
biological sample (e.g. using FISH or Comparative Genomic
Hybridization (CGH)). In another embodiment, the method can involve
measuring the level of MT-SP1 mRNA in the biological sample,
wherein an increased level of MT-SP1 RNA in the sample compared to
MT-SP1 RNA in a control sample indicates the presence (or
significant probability of the presence) of the cancer. The mRNA
determination can involve hybridizing (e.g. using a Northern blot,
a Southern blot, an array hybridization, an affinity
chromatography, an in situ hybridization, etc.) the mRNA to one or
more probes that specifically hybridize (under stringent
conditions) to a nucleic acid encoding the MT-SP1 protein. A probe
used in such measurements can optionally include a plurality of
probes that form an array of probes. Preferred detection methods
involve quantifying MT-SP1 mRNA. In still another embodiment, the
level of MT-SP1 mRNA is measured using a nucleic acid amplification
reaction. In addition, or alternatively, the method can involve
determining the level (e.g. via a method selected from the group
consisting of capillary electrophoresis, a Western blot, mass
spectroscopy, ELISA, immunochromatography, and
immunohistochemistry) or activity of an MT-SP1 protein in the
biological sample. Preferred biological samples for these assays
include, but are not limited to excised tissue, whole blood, serum,
plasma, buccal scrape, saliva, cerebrospinal fluid, and urine.
[0015] In certain embodiments, it is desired to pre-screen test
agents for the ability to bind to an MT-SP1 nucleic acid and/or
protein. Such pre-screening methods typically involve (a)
contacting a nucleic acid encoding an MT-SP1 serine protease or an
MT-SP1 serine protease protein with a test agent; and (b) detecting
specific binding of the test agent to the MT-SP1 protein or nucleic
acid. Preferred test agents do not include antibodies, and/or
nucleic acids. In particularly preferred assay formats the MT-SP1
nucleic acid and/or protein is immobilized on a solid support,
while in other preferred assay formats, the test agent is
immobilized (e.g. in a 96 well plate, etc.). Preferred methods of
detecting binding utilize detectable labels (e.g. fluorescent
labels) and a particular preferred detection methods utilizes
fluorescent resonance energy transfer (FRET).
[0016] MT-SP1 levels are also good prognostic indicators for
various cancers as described herein. This invention therefore also
provides methods (prognostic assays) for evaluating the severity or
outcome of a cancer (e.g. for estimating length of survival of a
cancer patient). The methods preferably involve (a) obtaining a
biological sample from a cancer patient having at least a
preliminary diagnosis of cancer; (b) measuring MT-SP1 in said
sample and comparing the sample MT-SP1 level to the MT-SP1 level in
normal healthy humans wherein a sample MT-SP1 level in excess of
MT-SP1 levels in normal healthy humans indicates a reduced survival
expectancy compared to patients with normal MT-SP1 level.
Particular embodiments include a preliminary diagnosis of prostate
cancer, a cancer of the digestive tract, a breast cancer, and/or a
urogential cancer. Preferred biological samples, include, but are
not limited to a primary tumor or a tissue affected by the cancer
(e.g. a tumor biopsy) and/or samples selected from the group
consisting of whole blood, plasma, serum, synovial fluid,
cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow
aspirate, pleural effusion, urine, or tumor tissue. As indicated
above, MT-SP1 can be evaluated by copy number of MT-SP1 genomic
DNA, MT-SP1 mRNA levels, levels of nucleic acid(s) derived from
MT-SP1 mRNA (e.g. cDNAs, RT-PCR products, etc.), MT-SP1 protein
levels and/or MT-SP1 activity levels. In a preferred embodiments
the level of MT-SP1 is measured by immunohistochemical staining of
cells comprising the biological sample (e.g. tumor tissue cells)
and/or via an immunoassay (e.g., ELISA using an anti-MT-SP1
antibody as described above).
[0017] Also provided are methods of treating a cancer in a patient.
The methods involve performing one or more of the prognostic assays
described herein on a cancer patient having at least a preliminary
diagnosis of a cancer; and (c) selecting a patient identified with
an MT-SP1 level excess of MT-SP1 levels in normal healthy humans
and providing an adjuvant cancer therapy (e.g. a therapy selected
from the group consisting of chemotherapy, radiation therapy,
reoperation, antihormone therapy, and immunotherapy).
[0018] This invention also affords methods of screening for
recurrence of a cancer after removal of a primary tumor. These
methods involve performing one or more of the assays described
herein on a biological sample from a cancer patient following
removal of a primary tumor. The assay can be repeated at a
multiplicity of instances after removal of the primary tumor.
[0019] Similarly the assays of this invention provide methods of
monitoring the effectiveness of cancer treatment in patients. This
involves obtaining a first biological sample from said patient
prior to or following one or more treatments of a cancer; obtaining
a second biological sample from said cancer patient during or after
said one or more treatments; evaluating the samples for MT-SP1
level as described herein where a lower level of MT-SP1 in the
second sample as compared to the MT-SP1 level in the first sample
indicates efficacy of the treatment(s). Typically the treatments
involve chemotherapy, radiation therapy, immunotherapy, anti
hormone therapy, or surgery.
[0020] It was also a discovery of this invention that MT-SP1
provides a good target for specifically delivering an effector
(e.g. a liposome, a cytotoxin, a detectable label) to a cell (e.g.
a tumor cell) expressing MT-SP1. The methods involve providing a
chimeric moiety comprising an effector (e.g. an effector molecule)
attached to an anti-MT-SP1 antibody; and contacting the cell with
said chimeric moiety whereby the chimeric moiety binds (e.g.
specifically binds) to the "target" cell. In preferred embodiments,
the cell (e.g. tumor cell) internalizes at least a portion (e.g. a
detectable or measurable amount) of the molecule. Preferred cells
include cancer cells, e.g., cells from a prostate cancer, a cancer
of the digestive tract, a breast cancer, and a urogential cancer.
Preferred effectors include a cytotoxin, a detectable label, a
radionuclide, a drug, a liposome, a ligand, and an antibody. In
particularly preferred embodiments, the chimeric moiety is a fusion
protein. Preferred fusion proteins include effectors selected from
the group consisting of ricin, abrin, Diphtheria toxin, and
Pseudomonas exotoxin. In other preferred embodiments, the effector
is cytotoxic and/or a liposome comprising an anti-cancer drug (e.g.
doxirubicin, vinblastine, vincristine, taxol, doxirubicin, and
genistein).
[0021] This invention also provides chimeric moieties (e.g.
chimeric molecules) comprising an effector attached to an
anti-MT-SP1 antibody. Preferred effectors include cytotoxin, a
detectable label, a radionuclide, a drug, a liposome, a ligand, and
an antibody. Preferred chimeric moieties are fusion proteins and
particularly preferred fusion proteins have cytotoxic effectors
(e.g., ricin, abrin, Diphtheria toxin, Pseudomonas exotoxin, etc.).
Other preferred effectors include a liposome comprising an
anti-cancer drug as described above. The chimeric moieties
described herein can be formulated as pharmaceutical compositions
comprising the chimeric moiety and pharmaceutically acceptable
excipient.
[0022] This invention also provides methods of impairing growth of
tumor cells expressing an MT-SP1 protein. The methods comprise
contacting the tumor cells with a chimeric molecule comprising an
anti-MT-SP1 antibody; and a cytotoxic effector as described
herein.
DEFINITIONS
[0023] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogs of natural
nucleotides that can function in a similar manner as naturally
occurring nucleotides.
[0024] A "nucleic acid derived from an mRNA transcript" or "nucleic
acid derived from an MT-SP1 gene" refers to a nucleic acid for
whose synthesis the mRNA transcript or a subsequence thereof or the
MT-SP1 gene or subsequence thereof has ultimately served as a
template. Thus, a cDNA reverse transcribed from an mRNA, an RNA
transcribed from that cDNA, a DNA amplified from the cDNA, an RNA
transcribed from the amplified DNA, etc., are all derived from the
mRNA transcript and detection of such derived products is
indicative of the presence and/or abundance of the original
transcript in a sample. Thus, suitable samples include, but are not
limited to, mRNA transcripts of the gene or genes, cDNA reverse
transcribed from the mRNA, cRNA transcribed from the cDNA, DNA
amplified from the genes, RNA transcribed from amplified DNA, and
the like.
[0025] The terms "isolated" "purified" or "biologically pure" refer
to material which is substantially or essentially free from
components which normally accompany it as found in its native
state.
[0026] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0027] An amino acid, identified by name herein "e.g., arginine" or
"arginine residue" as used herein refers to natural, synthetic, or
version of the amino acids. Thus, for example, an arginine can also
include arginine analogs that offer the same or similar
functionality as natural arginine with respect to their ability of
be incorporated into a polypeptide, effect folding of that
polypeptide and effect interactions of that polypeptide with other
polypeptide(s).
[0028] The phrase "nucleic acid encoding" or "nucleic acid sequence
encoding" refers to a nucleic acid that directs the expression of a
specific protein or peptide. The nucleic acid sequences include
both the DNA strand sequence that is transcribed into RNA and the
RNA sequence that is translated into protein. The nucleic acid
sequences include both full-length nucleic acid sequences as well
as shorter sequences derived from the full-length sequences. It is
understood that a particular nucleic acid sequence includes the
degenerate codons of the native sequence or sequences which may be
introduced to provide codon preference in a specific host cell. The
nucleic acid includes both the sense and antisense strands as
either individual single strands or in the duplex form.
[0029] The term "MT-SP1" protease, as used herein refers to either
the membrane type serine protease exemplified (e.g. by SEQ ID NOs:
1 and 2) or to the class of serine proteases characterized by the
presence of a non-cleaved signal/anchor domain that anchors the
serine protease in the cell membrane (see, e.g., Parks & Lamb
(1991) Cell 64: 777-787; Parks & Lamb (193) J. Biol. Chem.,
268: 19101-19109). Typically, charged residues flank the sides of
the signal/anchor domain was analyzed. Charged residues on the
N-terminal side of the signal/anchor are important for proper
topology, while addition of charges to the C-terminal side of the
signal/anchor has little effect upon orientation. Removal of any of
the positive charges preceding the signal anchor led to partial
inversion of the topology, suggesting that each positive charge
contributes to the signal. These results indicate that a type II
membrane protein is characterized by a protein that lacks a
cleavable signal sequence (1) and has positively charged residues
on the N-terminal side of a long stretch of hydrophobic amino acids
(see, e.g., Walter and Lingappa (1986) Annu. Rev. Cell Biol. 2:
499-516).
[0030] The term "mutation", when used in reference to a polypeptide
refers to the change of one or more amino acid residues in a
polypeptide to residues other than those found in the "native" or
"reference (pre-mutation) form of that polypeptide. Mutations
include amino acid substitutions as well as insertions and/or
deletions. A mutation does not require that the particular amino
acid substitution or deletion be made to an already formed
polypeptide, but contemplates that the "mutated" polypeptide can be
synthesized de novo, e.g. through chemical synthesis or recombinant
means. It will be appreciated that the mutation can include
replacement of a natural amino acid with an "unnatural" amino
acid.
[0031] The term "prognostic" or "prognostic assay" refers to an
assay that provides an indication as to the outcome of a disease. A
prognostic assay need not indicate the presence or absence of a
disease. A negative prognostic assay might indicate the need for a
more aggressive therapeutic regimen.
[0032] A "protease" is a polypeptide that cleaves another
polypeptide at a particular site (amino acid sequence). The
protease can also be self-cleaving.
[0033] A protease is said to be "specific" for another polypeptide
when it characteristically cleaves the other "substrate"
polypeptide at a particular amino acid sequence. The specificity
can be absolute or partial (i.e., a preference for a particular
amino acid or amino acid sequence).
[0034] The term "specifically binds" when used to refer to binding
proteins herein indicates that the binding preference (e.g.,
affinity for the target molecule/sequence is at least 2 fold, more
preferably at least 5 fold, and most preferably at least 10 or 20
fold over a non-specific (e.g. randomly generated molecule lacking
the specifically recognized amino acid or amino acid sequence)
target molecule.
[0035] The term "phage", when used in the context of polypeptide
display, includes bacteriophage as well as other "infective
viruses", e.g. viruses capable of infecting a mammalian, or other,
cell.
[0036] The term "chymotrypsin fold" refers to the anti-parallel
beta barrel protein "fold" characteristic of trypsin, chymotrypsin,
elastase, and related serine proteases (see, e.g., Branden and
Tooze (1991) Introduction to Protein Structure, Garland Publishing,
New York; Creighton (1993) Proteins, 2nd edition, W.H. Freeman
& Co., New York; Schulz and Schirmer (1979) Principles of
Protein Structure, Springer-Verlag, New York; Perutz (1992) Protein
Structure--New Approaches to Disease and Therapy, W.H. Freeman
& Co., New York; Fersht (1976) Enzyme Structure and Mechanism,
2nd ed., W.H. Freeman & Co., New York).
[0037] A "protease substrate" is a polypeptide that is specifically
recognized and cleaved by a protease.
[0038] The term "modulate" when used with respect to protease
activity refers to an alteration in the rate of reaction (protein
hydrolysis) catalyzed by a protease. An increase in protease
activity results in an increase in the rate of substrate hydrolysis
at a particular protease concentration and a protease modulator
that produces such an increase in protease activity is referred to
as an "activator" or "protease agonist". The terms "activator" or
"agonist" are thus used synonymously. A decrease in protease
activity refers to a decrease in the rate of substrate hydrolysis
at a particular protease concentration. Such a decrease may involve
total elimination of protease activity. A protease modulator that
produces a decrease in protease activity is referred to as a
"protease inhibitor". It will be appreciated that generally the
increase or decrease is as compared to the protease absent the
protease modulator.
[0039] The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof which specifically bind and recognize an analyte
(antigen). The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. An exemplary immunoglobulin (antibody) structural
unit comprises a tetramer. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) refer to these light and heavy chains
respectively.
[0040] Antibodies exist e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Fundamental Immunology, Third Edition, W. E.
Paul, ed., Raven Press, N.Y. 1993). While various antibody
fragments are defined in terms of the digestion of an intact
antibody, one of skill will appreciate that such fragments may be
synthesized de novo either chemically or by utilizing recombinant
DNA methodology. Thus, the term antibody, as used herein, also
includes antibody fragments either produced by the modification of
whole antibodies, those synthesized de novo using recombinant DNA
methodologies (e.g., single chain Fv), and those found in display
libraries (e.g. phage display libraries).
[0041] The phrases "hybridizing specifically to" or "specific
hybridization" or "selectively hybridize to", refer to the binding,
duplexing, or hybridizing of a nucleic acid molecule preferentially
to a particular nucleotide sequence under stringent conditions when
that sequence is present in a complex mixture (e.g., total
cellular) DNA or RNA.
[0042] The term "stringent conditions" refers to conditions under
which a probe will hybridize preferentially to its target
subsequence, and to a lesser extent to, or not at all to, other
sequences. "Stringent hybridization" and "stringent hybridization
wash conditions" in the context of nucleic acid hybridization
experiments such as Southern and northern hybridizations are
sequence dependent, and are different under different environmental
parameters. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes part I chapter 2 Overview of principles of hybridization and
the strategy of nucleic acid probe assays, Elsevier, New York.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5 C lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Very stringent conditions are selected to
be equal to the T.sub.m for a particular probe.
[0043] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formamide with 1 mg of heparin at 42 C, with the
hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15 M NaCl at 72 C for about 15
minutes. An example of stringent wash conditions is a 0.2.times.SSC
wash at 65 C for 15 minutes (see, Sambrook et al. (1989) Molecular
Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) supra
for a description of SSC buffer). Often, a high stringency wash is
preceded by a low stringency wash to remove background probe
signal. An example medium stringency wash for a duplex of, e.g.,
more than 100 nucleotides, is 1.times.SSC at 45 C for 15 minutes.
An example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6.times.SSC at 40 C for 15 minutes. In general, a
signal to noise ratio of 2.times. (or higher) than that observed
for an unrelated probe in the particular hybridization assay
indicates detection of a specific hybridization. Nucleic acids
which do not hybridize to each other under stringent conditions are
still substantially identical if the polypeptides which they encode
are substantially identical. This occurs, e.g., when a copy of a
nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code.
[0044] In one particularly preferred embodiment, stringent
conditions are characterized by hybridization in 1 M NaCl, 10 mM
Tris-HCl, pH 8.0, 0.01% Triton X-100, 0.1 mg/ml fragmented herring
sperm DNA with hybridization at 45.degree. C. with rotation at 50
RPM followed by washing first in 0.9 M NaCl, 0.06 M
NaH.sub.2PO.sub.4, 0.006 M EDTA, 0.01% Tween-20 at 45.degree. C.
for 1 hr, followed by 0.075 M NaCl, 0.005 M NaH.sub.2PO.sub.4, 0.5
mM EDTA at 45.degree. C. for 15 minutes.
[0045] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0046] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 60%, preferably 80%, most
preferably 90-95% nucleotide or amino acid residue identity, when
compared and aligned for maximum correspondence, as measured using
one of the following sequence comparison algorithms or by visual
inspection. Preferably, the substantial identity exists over a
region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably the sequences are substantially
identical over at least about 150 residues. In a most preferred
embodiment, the sequences are substantially identical over the
entire length of the coding regions.
[0047] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0048] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman (1988)
Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by visual inspection (see generally
Ausubel et al., supra).
[0049] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle (1987) J. Mol. Evol. 35:351-360. The method
used is similar to the method described by Higgins & Sharp
(1989) CABIOS 5: 151-153. The program can align up to 300
sequences, each of a maximum length of 5,000 nucleotides or amino
acids. The multiple alignment procedure begins with the pairwise
alignment of the two most similar sequences, producing a cluster of
two aligned sequences. This cluster is then aligned to the next
most related sequence or cluster of aligned sequences. Two clusters
of sequences are aligned by a simple extension of the pairwise
alignment of two individual sequences. The final alignment is
achieved by a series of progressive, pairwise alignments. The
program is run by designating specific sequences and their amino
acid or nucleotide coordinates for regions of sequence comparison
and by designating the program parameters. For example, a reference
sequence can be compared to other test sequences to determine the
percent sequence identity relationship using the following
parameters: default gap weight (3.00), default gap length weight
(0.10), and weighted end gaps.
[0050] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al. (1990)
J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses
is publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.
Acad. Sci. USA 89:10915).
[0051] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0052] The term "biological sample" refers to sample is a sample of
biological tissue, cells, or fluid that, in a healthy and/or
pathological state, contains a nucleic acid or polypeptide that is
to be detected according to the assays described herein. Such
samples include, but are not limited to, cultured cells, primary
cell preparations, sputum, amniotic fluid, blood, tissue or fine
needle biopsy samples, urine, peritoneal fluid, and pleural fluid,
or cells therefrom. Biological samples may also include sections of
tissues (e.g, frozen sections taken for histological purposes).
Although the sample is typically taken from a human patient, the
assays can be used to detect MT-SP1 in samples from any mammal,
such as dogs, cats, sheep, cattle, and pigs, etc. The sample may be
pretreated as necessary by dilution in an appropriate buffer
solution or concentrated, if desired. Any of a number of standard
aqueous buffer solutions, employing one of a variety of buffers,
such as phosphate, Tris, or the like, at physiological pH can be
used.
[0053] The term "test agent" refers to an agent that is to be
screened in one or more of the assays described herein. The agent
can be virtually any chemical compound. It can exist as a single
isolated compound or can be a member of a chemical (e.g.
combinatorial) library. In a particularly preferred embodiment, the
test agent will be a small organic molecule.
[0054] The term "effector" molecule refers to one or more molecules
comprising a "chimeric molecule or chimeric moiety" whose
"activity" it is desired to deliver (into, adjacent to or in the
proximity of) a target cell or cells. The activity need not be
activity on the cell, but can simply provide a property (e.g.
detectability by x-rays, elevated radiosensitivity, etc.) not
normally present at or in the cell. The effector while often a
single molecule also encompases multi-molecular entities (e.g.
liposomes containing drugs, etc.). It is also recognized that one
or more anti-MT-SP1 antibodies may be attached to any effector or,
conversely, one or more effectors can be attached to a single
MT-SP1 antibody.
[0055] The term "anti-cancer" drug is used herein to refer to one
or a combination of drugs conventionally used to treat cancer. Such
drugs are well known to those of skill in the art and include, but
are not limited to doxirubicin, vinblastine, vincristine, taxol,
etc.
[0056] The term "small organic molecules" refers to molecules of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da.
[0057] The term "conservative substitution" is used in reference to
proteins or peptides to reflect amino acid substitutions that do
not substantially alter the activity (specificity or binding
affinity) of the molecule. Typically conservative amino acid
substitutions involve substitution one amino acid for another amino
acid with similar chemical properties (e.g. charge or
hydrophobicity). The following six groups each contain amino acids
that are typical conservative substitutions for one another: 1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan
(W).
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 shows the nucleotide sequence of the cDNA encoding
human MT-SP1 (SEQ ID NO: 1) and predicted protein sequence (SEQ ID
NO: 2). Numbering indicates nucleotide or amino acid residue. Amino
acids are shown in single-letter code. The termination codon is
shown by an asterisk (*). The underlined stop codon at nucleotide
10 is in frame with the initiating methionine. The Kozak consensus
sequence (Kozak (1991) J. Cell Biol. 115: 887-903) at the start
codon is underlined at nucleotide 32. The predicted N-glycosylation
sites at amino acids 109, 302, 485, and 772 are underlined. A
possible polyadenylation sequence (Nevins (1983) Ann. Rev. Biochem.
52: 441-466) at nucleotide 3120 is also underlined. The catalytic
triad in the serine protease domain is highlighted: His656, Asp711
and Ser805.
[0059] FIG. 2, lane 1 shows the PCR products obtained using
degenerate primers designed from the consensus sequences flanking
the catalytic histidine (5' His-primer) and the catalytic serine
(3' Ser-primer). The products remaining between 400 and 550 bp
after digestion with BamHI were reamplified using the same
degenerate primers. The products from this second PCR are shown in
lane 2.
[0060] FIG. 3 shows the domain structure of human MT-SP1 compared
with the domain structure of hepsin (Leytus et al. (1988)
Biochemistry 27: 1067-1074) and enteropeptidase (Kitamoto et al.
(1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592). SA represents a
possible signal anchor, CUB represents a repeat first identified in
complement components C1r and C1s, the urchin embryonic growth
factor and bone morphogenetic protein 1 (Bork and Beckmann (1993)
J. Mol. Biol. 231: 539-545), L represents low-density lipoprotein
receptor repeat (Krieger and Herz (1994) Annu. Rev. Biochem. 63:
601-637), SP represents a chymotrypsin family serine protease
domain (Perona and Craik (1997) J. Biol. Chem. 272: 29987-29990),
MAM represents a domain homologous to members of a family defined
by meprin, protein A5, and the protein tyrosine phosphatase .mu.
(Beckmann and Bork (1993) Trends Biochem. Sci. 18: 40-41), and MSCR
represents a macrophage scavenger receptor cysteine-rich motif
(Krieger and Herz (1994) Annu. Rev. Biochem. 63: 601-637). The
predicted disulfide linkages are shown labeled as C-C.
[0061] FIGS. 4A, 4B, and 4C show multiple sequence alignments of
MT-SP1 structural motifs. L represent loops, represent beta sheets,
represent alpha helices, and S-S represent disulfides. FIG. 4A
shows multiple sequence alignment of the serine protease domain of
MT-SP1 with human trypsinogen B (Emi et al. (1986) Gene 41:
305-310), human enterokinase (Kitamoto et al. (1995) Biochemistry
34: 4562-4568), human hepsin (Leytus et al. (1988) Biochemistry 27:
1067-1074), human tryptase 2 (Vanderslice et al. (1990) Proc. Natl.
Acad. Sci. USA 87: 3811-3815), and human chymotrypsinogen 13
(Tomita et al. (1989) Biochem. Biophys. Res. Commun. 158: 569-575),
using standard chymotrypsin numbering. Conserved catalytic and
structural residues described in the text are underlined. FIG. 4B
shows alignment of MT-SP1 LDLR with domains of the LDL receptor
(Sudhof et al. (1985) Science 228: 815-822). FIG. 4C shows
alignment of the CUB domains of MT-SP1 with those found in human
enterokinase (Kitamoto et al. (1995) Biochemistry 34: 4562-4568),
human bone morphogenetic protein 1 (Wozney et al. (1988) Science
242: 1528-1534), and complement component C1R (Leytus et al. (1986)
Biochemistry 25: 48554863).
[0062] FIGS. 5A and 5B show the tissue distribution of MT-SP1 mRNA
levels. Northern blots of human poly(A)+ RNA from assorted human
tissues was hybridized with radiolabeled cDNA probes as described
under Materials and Methods. The upper panel shows hybridization
using a MT-SP1 1.3 kB cDNA fragment derived from EST clone w39209
and exposed overnight. The lower panel shows the same blot after
being stripped and rehybridized with a loading standard (FIG. 5A)
.beta.-actin or (FIG. 5B) human glyceraldehyde phosphate
dehydrogenase (GAPDH) cDNA probe exposed for two hours. The
mobility of RNA size standards are indicated at the left.
[0063] FIGS. 6A and 6B show activation and purification of
His-tagged MT-SP1 protease domain. A representative experiment is
shown in (FIG. 6A) and (FIG. 6B). FIG. 6A: Activation at 4.degree.
C. is monitored using SDS-PAGE. The upper band represents
inactivated protease domain, and the lower band represents active
protease (also verified by N-terminal sequencing). FIG. 6B: The
activation of the protein was monitored using
hexahydrotyrosyl-glycyl-arginyl-paranitroanilide as a synthetic
substrate for the protease domain. (C) Inactive Ser.sup.805 Ala
protease domain is cleaved with 10 nM activated His-tagged MT-SP1
protease domain at 37.degree. C. The specific cleavage of active
MT-SP1 protease domain is required for proper processing at the
activation site. Active protease domain is shown in lane 7 (+), and
no cleavage of the untreated inactive protease domain is observed
(lane 8, -).
DETAILED DESCRIPTION
[0064] This invention pertains to the discovery of novel
membrane-type serine proteases whose inhibition results in
inhibition of mouse and rat prostate differentiation and the
retardation of growth of human PC-3 TRAMP prostatic cancer cells.
The prototypical protease of this invention is referred to herein
as membrane-type serine protease 1 (MT-SP1).
[0065] The cloning and characterization of the MT-SP1 cDNA showed
that it encodes a mosaic protein that contains a transmembrane
signal anchor, two CUB domains, four LDLR repeats, and a serine
protease domain. Northern blotting showed broad expression of
MT-SP1 in a variety of epithelial tissues with high levels of
expression in the human gastrointestinal tract and the prostate. In
particular MT-SP1 showed significant expression in the endothelium
of tumor blood vessels
[0066] The data presented herein indicate that expression of the
MT-SP1 membrane-type serine protease(s) are associated with the
presence of, or proclivity to, cancer. In particular, without being
bound to a theory, it is believed that the membrane-type serine
protease MT-SP1 participates in a proteolytic cascade that results
in cell growth and or differentiation. Another structurally similar
membrane-type serine protease, enteropeptidase (FIG. 3), is
involved in a proteolytic cascade by which activation of
trypsinogen leads to activation of downstream intestinal proteases
(Huber and Bode (1978) Acc. Chem. Res. 11: 114-122).
Enteropeptidase is expressed only in the enterocytes of the
proximal small intestine thus precisely restricting activation of
trypsinogen. Thus, in contrast to secreted proteases that may
diffuse throughout the organism, the membrane association of MT-SP1
allows the proteolytic activity to be precisely localized, which
may is important for proper physiological function. Improper
localization of the enzyme or levels of downstream substrates could
lead to disease.
[0067] We have found subcutaneous coinjection of PC-3 cells with
wild-type ecotin or ecotin M84R/M85R led to a decrease in the
primary tumor size compared to animals in whom PC-3 cells and
saline were injected. Since wild-type ecotin is a poor, micromolar
inhibitor of uPA, serine proteases other than uPA (e.g., an MT-SP1)
are believed to be involved in this primary tumor proliferation.
Both wild-type ecotin and ecotin M84R/M85R are potent, subnanomolar
inhibitors of MT-SP1, strongly suggesting that MT-SP1 plays an
important role in progression of epithelial cancers expressing this
protease.
[0068] In addition, MT-SP1 is associated with tumors (e.g. is a
good tumor marker). In particular, immunohistochemical examination
of gastric cancer tissue revealed MT-SP1 expression in cancer
cells, endothelial cells and some leukocytes. In these tissues,
endothelial cells showed especially intensive MT-SP1
immunoreactivity indicating that MT-SP1 might play an important
role in vascular cells particularly in angiogenesis of tumor and
tumor-related blood vessels.
[0069] In addition, overall survival for groups of gastric
carcinoma patients with highly MT-SP1 expressing endothelium
revealed poor prognosis compared to those with low or no MT-SP1.
Higher MT-SP1 expression in endothelium was significantly
associated with lower survival rate.
[0070] MT-SP1 thus appears to be a good diagnostic, prognostic, or
therapeutic target. In one embodiment, this invention therefore
provides methods of screening for (e.g. diagnosing) the presence or
absence of a cancer by detecting the level of MT-SP1 expression
and/or activity. In a particularly preferred embodiment this
invention provides methods of screening for (e.g. diagnosing) the
presence of a metastatic cell.
[0071] In another embodiment, this invention provides prognostic
methods, e.g., methods of estimating length of survival of a cancer
patient, or evaluating the severity of disease or the likelihood of
disease recurrence in a cancer patient. These methods also involve
determining the level of MT-SP1 expression and/or activity, where
higher expression levels indicate greater disease severity, poorer
outcome or greater likelihood of disease recurrence.
[0072] MT-SP1 also provides a convenient diagnostic tag for
localizing a tumor or cancer cell. This involves providing
anti-MT-SP1 antibodies attached to a detectable tag (e.g.
radioactive or radiopaque tage). The labeled MT-SP1 antibody will
localize on the surface of tumor cells expressing MT-SP1 and
detection of the tag provides an indication for the presence,
locality, and size of the cancer.
[0073] Having identified a novel protease involved with the
etiology of cancers, particularly invasive cancers, the MT-SP1 DNA,
mRNA, and protein product provide good targets for the action of
putative modulators. This invention thus, also provides methods of
screening for modulators of MT-SP1 expression and/or activity. In
addition simple binding assays can be used to identify agents (e.g.
small organic molecules, antisense molecules, ribozymes,
antibodies, etc.) that interact specifically with the MT-SP1
nucleic acids and/or proteins. Screening agents for such specific
binders identifies putative agents likely to modulate MT-SP1
expression and/or activity. Collections of such agents provide
"biases" molecular libraries that are useful candidates for a
variety of screening systems.
[0074] The localization of MT-SP1 in tumors, and in particular, on
vascular endothelial cells associated with tumors provides a
convenient method for specifically delivering an effector (e.g. an
effector molecule) to such cells. The method involves attaching one
or more molecules that specifically bind to MT-SP1 (e.g. an
anti-MT-SP1 antibody) to the effector it is desired to deliver and
administering the composition to the subject (e.g.
intraperioneally, intravenously, direct injection into tumor site,
etc.). The chimeric antibody-effector composition will localize at
the tumor site or on cells (e.g. tumor or metastatic cells
expressing MT-SP1).
[0075] This invention also provides MT-SP1 nucleic acids and
isolated (e.g. recombinantly expressed) proteins. Such isolated
proteins are useful specific proteases in their own right. In
addition they can be used to help "dissect" metabolic pathways
and/or can be used as effective immunogens to raise anti-MT-SP1
antibodies.
I. Nucleic Acids Encoding Membrane-Type Serine Proteases.
[0076] Using the information provided herein, (e.g. MT-SP1 cDNA
sequence, primers, etc.) the nucleic acids (e.g., encoding full
length MT-SP1 or MT-SP1 proteolytic domain or other subsequences of
the MT-SP1 cDNA, genomic DNA, mRNA, etc). are prepared using
standard methods well known to those of skill in the art. For
example, the MT-SP1 nucleic acid(s) may be cloned, or amplified by
in vitro methods, such as the polymerase chain reaction (PCR), the
ligase chain reaction (LCR), the transcription-based amplification
system (TAS), the self-sustained sequence replication system (SSR),
etc. A wide variety of cloning and in vitro amplification
methodologies are well-known to persons of skill. Examples of these
techniques and instructions sufficient to direct persons of skill
through many cloning exercises are found in Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology 152
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
(Sambrook et al.); Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel); Cashion et al., U.S. Pat. No.
5,017,478; and Carr, European Patent No. 0,246,864. Examples of
techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, Sambrook, and Ausubel,
as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR
Protocols A Guide to Methods and Applications (Innis et al. eds)
Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim &
Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research
(1991) 3: 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:
1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874;
Lomell et al. (1989) J. Clin. Chem., 35:1826; Landegren et al.,
(1988) Science, 241: 1077-1080; Van Brunt (1990) Biotechnology, 8:
291-294; Wu and Wallace, (1989) Gene, 4: 560; and Barringer et al.
(1990) Gene, 89: 117.
[0077] The isolation and expression of an MT-SP1 nucleic acid is
illustrated in Example 1. In one preferred embodiment, the MT-SP1
cDNA can be isolated by routine cloning methods. The cDNA sequence
provided in SEQ ID NO: 1 can be used to provide probes that
specifically hybridize to the MT-SP1 gene, in a genomic DNA sample,
or to the MT-SP1 mRNA, in a total RNA sample (e.g., in a Southern
blot). Once the target MT-SP1 nucleic acid is identified (e.g., in
a Southern blot), it can be isolated according to standard methods
known to those of skill in the art (see, e.g., Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3,
Cold Spring Harbor Laboratory; Berger and Kimmel (1987) Methods in
Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San
Diego: Academic Press, Inc.; or Ausubel et al. (1987) Current
Protocols in Molecular Biology, Greene Publishing and
Wiley-Interscience, New York). Methods of screening human cDNA
libraries for the MT-SP1 are provided in Example 1.
[0078] In another preferred embodiment, the human MT-SP1 cDNA can
be isolated by amplification methods such as polymerase chain
reaction (PCR). In a preferred embodiment, the MT-SP1 sequence is
amplified from a cDNA sample (e.g., double stranded placental cDNA
(Clontech)) using the primers routinely derived from the sequence
illustrated in FIG. 1 (SEQ. ID NO: 1). Preferred primers are primer
1, nucleotides 37-54 of SEQ ID NO: 1) and primer 2, nucleotides
2604-2583 of the complement of SEQ ID NO: 1. Preferred
amplification conditions include 30 cycles of 1 minute denaturing
at 94.degree. C., 1 minute annealing at 54.degree. C., 3 minutes of
extension at 72.degree. C., followed by a final 15 minute extension
at 72.degree. C. Preferred template includes full length PCIII
cDNA.
[0079] Where the MT-SP1 gDNA, cDNA, mRNA or their subsequences are
to be used as nucleic acid probes, it is often desirable to label
the nucleic acids with detectable labels. The labels may be
incorporated by any of a number of means well known to those of
skill in the art. However, in one preferred embodiment, the label
is simultaneously incorporated during the amplification step in the
preparation of the sample nucleic acids. Thus, for example,
polymerase chain reaction (PCR) with labeled primers or labeled
nucleotides will provide a labeled amplification product. In
another preferred embodiment, transcription amplification using a
labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP)
incorporates a label into the transcribed nucleic acids.
[0080] Alternatively, a label may be added directly to an original
nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the
amplification product after the amplification is completed. Means
of attaching labels to nucleic acids are well known to those of
skill in the art and include, for example nick translation or
end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic
acid and subsequent attachment (ligation) of a nucleic acid linker
joining the sample nucleic acid to a label (e.g., a fluorophore).
Suitable labels are described below.
II. Cloning and Expression of Membrane-Type Serine Proteases.
[0081] It is often desirable to provide isolated membrane-type
serine proteases of this invention (e.g., MT-SP1). These
polypeptides can be used to raise an immune response and thereby
generate antibodies specific to the intact MT-SP1 or to various
subsequences or domains thereof. As explained below, the MT-SP1
polypeptides and various fragments thereof can be conveniently
produced using synthetic chemical syntheses or recombinant
expression methodologies. In addition to the intact full-length
MT-SP1 polypeptide, in some embodiments, it is often desirably to
express immunogenically relevant fragments (e.g. fragments that can
be used to raise specific anti-MT-SP1 antibodies). In other
preferred embodiments, the protein is expressed as an inactive form
(a zymogen or pro-enzyme) that is activated, e.g. via autocleavage,
or alternatively, the enzymatic (proteolytic) domain can be
expressed alone.
[0082] A) De Novo Chemical Synthesis.
[0083] The MT-SP1 serine protease precursors, the catalytic domain
(active protease), or other subsequences of the MT-SP1
polypeptide(s) may be synthesized using standard chemical peptide
synthesis techniques. Where the desired subsequences are relatively
short (e.g., when a particular antigenic determinant is desired)
the molecule may be synthesized as a single contiguous polypeptide.
Where larger molecules are desired, subsequences can be synthesized
separately (in one or more units) and then fused by condensation of
the amino terminus of one molecule with the carboxyl terminus of
the other molecule thereby forming a peptide bond.
[0084] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2:
Special Methods in Peptide Synthesis, Part A., Merrifield, et al.
(1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al. (1984)
Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford,
Ill.
[0085] B) Recombinant Expression.
[0086] In a preferred embodiment, the MT-SP1 proteins or
subsequences thereof (e.g. proteolytic domain), are synthesized
using recombinant expression systems. Generally this involves
creating a DNA sequence that encodes the desired protein, placing
the DNA in an expression cassette under the control of a particular
promoter, expressing the protein in a host, isolating the expressed
protein and, if required, renaturing the protein.
[0087] DNA encoding the MT-SP1 proteins described herein can be
prepared by any suitable method as described above, including, for
example, cloning and restriction of appropriate sequences or direct
chemical synthesis by methods such as the phosphotriester method of
Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester
method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the
diethylphosphoramidite method of Beaucage et al. (1981) Tetra.
Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No.
4,458,066.
[0088] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0089] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0090] In one embodiment, the MT-SP1 nucleic acids of this
invention can be cloned using DNA amplification methods such as
polymerase chain reaction (PCR) (see, e.g., Example 1). Thus, for
example, the nucleic acid sequence or subsequence is PCR amplified,
using a sense primer containing one restriction site (e.g., NdeI)
and an antisense primer containing another restriction site (e.g.,
HindIII). This will produce a nucleic acid encoding the desired
MT-SP1 sequence or subsequence and having terminal restriction
sites. This nucleic acid can then be easily ligated into a vector
containing a nucleic acid encoding the second molecule and having
the appropriate corresponding restriction sites. Suitable PCR
primers can be determined by one of skill in the art using the
sequence information provided in SEQ ID NOs:1 and 2 and
representative primers are provided herein. Appropriate restriction
sites can also be added to the nucleic acid encoding the MT-SP1
protein or protein subsequence by site-directed mutagenesis. The
plasmid containing the MT-SP1 sequence or subsequence is cleaved
with the appropriate restriction endonuclease and then ligated into
the vector encoding the second molecule according to standard
methods.
[0091] The nucleic acid sequences encoding MT-SP1 proteins or
protein subsequences may be expressed in a variety of host cells,
including E. coli, other bacterial hosts, yeast, and various higher
eukaryotic cells such as the COS, CHO and HeLa cells lines and
myeloma cell lines. The recombinant protein gene will be operably
linked to appropriate expression control sequences for each host.
For E. coli this includes a promoter such as the T7, trp, or lambda
promoters, a ribosome binding site and preferably a transcription
termination signal. For eukaryotic cells, the control sequences
will include a promoter and often an enhancer (e.g., an enhancer
derived from immunoglobulin genes, SV40, cytomegalovirus, etc.),
and a polyadenylation sequence, and may include splice donor and
acceptor sequences.
[0092] The plasmids of the invention can be transferred into the
chosen host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0093] Once expressed, the recombinant MT-SP1 protein(s) can be
purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like (see, generally,
R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.;
Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein
Purification, Academic Press, Inc. N.Y.). Substantially pure
compositions of at least about 90 to 95% homogeneity are preferred,
and 98 to 99% or more homogeneity are most preferred. Once
purified, partially or to homogeneity as desired, the polypeptides
may then be used (e.g., as immunogens for antibody production). The
cloning and expression of a MT-SP1 polypeptides is illustrated in
Example 1.
[0094] In a preferred embodiment, the MT-SP1 nucleic acid(s) are
transformed into E. coli X-90 to afford high-level expression of
recombinant protease gene products (Evnin et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 6659-6663). Expression and purification of the
recombinant enzyme from solubilized inclusion bodies is performed
according to the method of Unal et al. (1997) J. Virol. 71,
7030-7038.
[0095] One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the MT-SP1
protein(s) may possess a conformation substantially different than
the native conformations of the constituent polypeptides. In this
case, it may be necessary to denature and reduce the polypeptide
and then to cause the polypeptide to re-fold into the preferred
conformation. Methods of reducing and denaturing proteins and
inducing re-folding are well known to those of skill in the art
(see, e.g., Debinski et al. (1993) J. Biol. Chem., 268:
14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:
581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).
Debinski et al., for example, describes the denaturation and
reduction of inclusion body proteins in guanidine-DTE. The protein
is then refolded in a redox buffer containing oxidized glutathione
and L-arginine.
[0096] One of skill would recognize that modifications can be made
to the MT-SP1 proteins without diminishing their biological
activity. Some modifications may be made to facilitate the cloning,
expression, or incorporation of the targeting molecule into a
fusion protein. Such modifications are well known to those of skill
in the art and include, for example, a methionine added at the
amino terminus to provide an initiation site, or additional amino
acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
III. Diagnostics/Prognostics--Assays of Membrane-Type Serine
Protease Level or Activity.
[0097] A) Diagnostic Applications.
[0098] MT-SP1 provides an effective marker for the
detection/diagnosis of a wide variety of cancers. Diagnosis of
disease based on measured levels of MT-SP1 can be made by
comparison to levels measured in a disease-free control group or
background levels measured in a particular patient. The diagnosis
can be confirmed by correlation of the assay results with other
signs of disease known to those skilled in the clinical arts, such
as the diagnostic standards for breast cancer, gastric cancer,
prostate cancer, etc. Because in certain instances serum MT-SP1 may
stem from sources other than the tissue of interest, in certain
embodiments, a sample is preferably taken from the tissue of
interest. However, as described below, in many instances basic
differential diagnosis allows identification of the pathology
resulting in elevated serum MT-SP1.
[0099] Particularly for the diagnosis and monitoring of cancers
(e.g., tumor metastasis), the preferred source for the assay sample
will be blood or blood products (e.g. plasma and/or serum) and/or
tissue biopsies. Those of ordinary skill in the art will be able to
readily determine which assay sample source is most appropriate for
use in diagnosis of a particular disease for which MT-SP1 is a
marker.
[0100] The levels of MT-SP1 that are indicative of the development
or amelioration of a particular cancer by disease and, to a lesser
extent, by patient. Appropriate background MT-SP1 levels in
particular tissues, pathologies, and patients or patient
populations or control populations can be determined by routine
screening according to standard methods well known to those of
skill in the art.
[0101] For purposes of diagnosing the onset, progression, or
amelioration of disease, variations in the levels of MT-SP1 of
interest will be those which differ by a statistically significant
level from the normal (i.e., healthy) population or from the level
measured in the same individual at a different time, and which
correlate to other clinical signs of disease occurrence and/or
prognosis and/or amelioration known to those skilled in the
clinical art pertaining to the disease of interest.
[0102] Thus, in general, any diagnosis or prognosis indicated by
MT-SP1 measurements made according to the methods of the invention
will be independently confirmed with reference to clinical
manifestations of disease known to practitioners of ordinary skill
in the clinical arts.
[0103] B) Prognostic Applications.
[0104] In prognostic applications, MT-SP1 levels are evaluated to
estimate the risk of recurrence of a cancer and thereby provide
information that facilitates the selection of treatment regimen.
Without being bound to a particular theory, it is believed that
tumors are heterogeneous (even within a particular tumor type, e.g.
colorectal cancer) with respect to elevated expression of MT-SP1.
Those tumor types resulting in elevated levels of MT-SP1 also show
a high likelihood of recurrence, e.g. after removal of a primary
tumor. Thus, measurement of MT-SP1 levels (before, during [i.e. in
blood or tissues removed during surgery], or after primary tumor
removal) provides a prognostic indication of the likelihood of
tumor recurrence. Where pathologies show elevated MT-SP1 levels
(e.g. as compared to those in normal healthy subjects) more
aggressive adjunct therapies (e.g. chemotherapy and/or
radiotherapy) may be indicated.
[0105] By way of further example, in gastric cancer stages III, IV,
patients (n=30) with an MT-SP LI of 40% or higher had a
significantly lower survival rate than those (n=11) without MT-SP1
expression in vascular cells of cancer tissues. In addition,
overall survival for groups of gastric carcinoma patients with
highly MT-SP1 expressing endothelium revealed poor prognosis
compared to those with low or no MT-SP1. Higher MT-SP1 expression
in endothelium was significantly associated with lower survival
rate indicating that MT-SP1 expression in endothelium around cancer
cells is an important prognostic factor in gastric cancer.
[0106] C) Evaluation of Treatment Efficacy.
[0107] The MT-SP1 markers of this invention can also be used to
evaluate treatment efficacy (e.g. amelioration of one or more
symptoms of a cancer). Where the amelioration of a disease (such as
cancer) can be related to reduction in levels of MT-SP1, MT-SP1
levels in a biological assay sample taken from the patient (e.g.,
blood) can be measured before (for background) and during or after
(e.g., at a designated time, periodically or randomly) the course
of treatment. Because reductions in MT-SP1 levels may be transient,
the assay will preferably be performed at regular intervals, (e.g.,
every 4 weeks, every 6 months, every year, etc.) closely before and
after each treatment. Depending on the course of treatment, tumor
load and other clinical variables, clinicians of ordinary skill in
the art will be able to determine an appropriate schedule for
performing the assay for diagnostic or disease/treatment monitoring
purposes.
[0108] Such monitoring methods can provide useful information to
guide a therapeutic regimen in a variety of contexts as explained
below.
[0109] 1) Checking for Recurrence of a Cancer.
[0110] In one embodiment, MT-SP1 is monitored simply to check for
the possible recurrence of a cancer after the primary tumor has
been removed. This method generally involves obtaining a biological
sample from a cancer patient following removal of a primary tumor;
and measuring the level of MT-SP1 in the sample. An elevated MT-SP1
level (e.g. as compared to the MT-SP1 level in normal healthy
humans) indicates a possible recurrence of a cancer. Where patients
have elevated MT-SP1 levels at the time of surgery, the subsequent
MT-SP1 monitoring is most informative after a period of time
sufficient to permit MT-SP1 levels to return to normal (e.g. about
34 weeks after surgery). Of course, monitoring can be performed
earlier to initiate tracking of changes in MT-SP1 levels. Where the
patient does not have an elevation in MT-SP1 at the time of surgery
increased MT-SP1 levels at any time after surgery indicate possible
recurrence of the cancer. Elevated levels can be evaluated relative
to levels in normal healthy people, or relative to MT-SP1 baseline
levels determined for the particular patient (e.g., prior to,
during, or immediately after surgery).
[0111] 2) Monitoring of Terminal Phase Patients.
[0112] In another embodiment, MT-SP1 monitoring can be used to
monitor the effectiveness of cancer treatment in patients with
elevated MT-SP1. Such monitoring is particularly useful in patients
in the terminal phase where the cancer has already metastasized so
that surgery will not completely eliminate the cancer. Such
patients will still be treated with radiation, chemotherapy, etc,
to give them additional months of survival (although in many cases
no cure). Periodic measurement of MT-SP1 provides the clinician
with a means of monitoring the progress of treatment.
[0113] 3) Checking the Efficacy of Surgical Removal of a Primary
Tumor.
[0114] In still another embodiment, MT-SP1 monitoring can be used
to check for the effectiveness of surgical removal of a primary
tumor, in those instances in which there is an elevation in MT-SP1
prior to surgery. Since our longitudinal study shows that removal
of the primary tumor causes the elevated MT-SP1 levels to fall to
normal, measurement of MT-SP1 in post operative blood (e.g., about
4 weeks after surgery) will reveal those instances in which surgery
did not remove all of the primary tumor, affected lymph nodes, and
any other metastasis sites.
[0115] D) Relevant Pathologies.
[0116] As indicated above, MT-SP1 provides an effective marker for
detection and/or evaluation of prognosis of a wide variety of
cancers including, but not limited to, gastric cancer, prostate
cancer, cancers of the urinary tract, lung cancer, bronchus cancer,
a colorectal cancer (cancer of the colon and/or rectum), breast
cancer, pancreas cancer, brain or central nervous system cancer,
peripheral nervous system cancer, esophageal cancer, cervical
cancer, melanoma, uterine or endometrial cancer, cancer of the oral
cavity or pharynx, liver cancer, kidney cancer, testes cancer,
biliary tract cancer, small bowel and appendix cancer, salivary
gland cancer, thyroid gland cancer, adrenal gland cancer, and
sarcomas such as osteosarcoma, chondrosarcoma, liposarcoma, and
malignant fibrous histiocytoma. In general, MT-SP1 is a
particularly good marker for metastatic cancers.
[0117] E) Relevant Pathologies.
[0118] As indicated above, MT-SP1 provides an effective marker for
detection and/or evaluation of prognosis of a wide variety of
cancers including, but not limited to, gastric cancer, prostate
cancer, cancers of the urinary tract, lung cancer, bronchus cancer,
a colorectal cancer (cancer of the colon and/or rectum), breast
cancer, pancreas cancer, brain or central nervous system cancer,
peripheral nervous system cancer, esophageal cancer, cervical
cancer, melanoma, uterine or endometrial cancer, cancer of the oral
cavity or pharynx, liver cancer, kidney cancer, testes cancer,
biliary tract cancer, small bowel and appendix cancer, salivary
gland cancer, thyroid gland cancer, adrenal gland cancer, and
sarcomas such as osteosarcoma, chondrosarcoma, liposarcoma, and
malignant fibrous histiocytoma. In general, MT-SP1 is a
particularly good marker for metastatic cancers.
IV. Assay Formats.
[0119] As indicated above, in one aspect, this invention is
premised on the discovery that membrane-type serine proteases (e.g.
MT-SP1) are associated with occurrence, growth, proliferation,
invasiveness, and angiogenesis of cancers. Thus, in one embodiment,
this invention provides methods of screening for cancers and/or
evaluating the severity of a cancer and/or the likelihood of
metastatic cells being present and/or developing and/or evaluating
the prognosis of a cancer. The methods involve detecting the
expression level and/or activity level of a membrane-type serine
protease where elevated expression levels and/or elevated activity
levels indicate the presence of a cancer and/or the presence of
invasive cancer cells and/or increased severity of the disease.
Thus, assays of copy number, expression level or level of activity
of one or MT-SP1 genes provides useful diagnostic and/or prognostic
information. Using the nucleic acid sequences and/or amino acid
sequences provided herein copy number and/or activity level can be
directly measured according to a number of different methods as
described below. In particular, expression levels of a gene can be
altered by changes in the copy number of the gene, and/or by
changes in the transcription of the gene product (i.e.
transcription of mRNA), and/or by changes in translation of the
gene product (i.e. translation of the protein), and/or by
post-translational modification(s) (e.g. protein folding,
glycosylation, etc.). Thus useful assays of this invention include
assaying for copy number, level of transcribed mRNA, level of
translated protein, activity of translated protein, etc. Examples
of such approaches are described below.
[0120] A) Sample Collection and Processing.
[0121] The MT-SP1 nucleic acid and/or protein is preferably
quantified in a biological sample derived from a mammal (e.g.,
whole blood, plasma, serum, synovial fluid, cerebrospinal fluid,
bronchial lavage, ascites fluid, bone marrow aspirate, pleural
effusion, urine, or tumor tissue), more preferably from a human
patient. Preferred biological samples include sample(s) of
biological tissue or fluid that contain MT-SP1 in a concentration
that may be correlated with the presence and/or prognosis of a
pathological state (e.g. a cancer). Particularly preferred
biological samples include, but are not limited to whole blood,
serum, plasma, synovial fluid, cerebrospinal fluid, bronchial
lavage, ascites fluid, pleural effusion, bone marrow aspirate,
urine, and tumor tissue.
[0122] The biological sample may be pretreated as necessary by
dilution in an appropriate buffer solution or concentrated, if
desired. Any of a number of standard aqueous buffer solutions,
employing one of a variety of buffers, such as phosphate, Tris, or
the like, at physiological pH can be used.
[0123] As indicated above, in a preferred embodiment, assays are
performed using whole blood, serum, or plasma or in tissue biopsies
and/or tissue sections. Obtaining and storing tissues, blood and/or
blood products are well known to those of skill in the art.
Typically blood is obtained by venipuncture. The blood may be
diluted by the addition of buffers or other reagents well known to
those of skill in the art and may be stored for up to 24 hours at
2-8.degree. C., or at -20.degree. C. or lower for longer periods,
prior to measurement of YKL-40. In a particularly preferred
embodiment, the blood or blood product (e.g. serum) is stored at
-70.degree. C. without preservative indefinitely.
[0124] B) Nucleic-Acid Based Assays.
[0125] 1) Target Molecules.
[0126] As indicated above, MT-SP1 gene expression can be varied by
changes in copy number of the gene and/or changes in the regulation
of gene expression. Changes in copy number are most easily detected
by direct changes in genomic DNA, while changes in expression level
can be detected by measuring changes in mRNA and/or a nucleic acid
derived from the mRNA (e.g. reverse-transcribed cDNA, etc.).
[0127] In order to measure the nucleic acid concentration in a
sample, it is desirable to provide a nucleic acid sample for such
analysis. In preferred embodiments the nucleic acid is found in or
derived from a biological sample. The term "biological sample", as
used herein, refers to a sample obtained from an organism or from
components (e.g., cells) of an organism. The sample may be of any
biological tissue or fluid. Frequently the sample will be a
"clinical sample" which is a sample derived from a patient. Such
samples include, but are not limited to, sputum, blood, tissue or
fine needle biopsy samples, urine, peritoneal fluid, and pleural
fluid, or cells therefrom. Biological samples may also include
sections of tissues such as frozen sections taken for histological
purposes.
[0128] The nucleic acid (either genomic DNA or mRNA) is, in certain
preferred embodiments, isolated from the sample according to any of
a number of methods well known to those of skill in the art. One of
skill will appreciate that where alterations in the copy number of
a gene are to be detected genomic DNA is preferably isolated.
Conversely, where expression levels of a gene or genes are to be
detected, preferably RNA (mRNA) is isolated.
[0129] Methods of isolating total mRNA are well known to those of
skill in the art. For example, methods of isolation and
purification of nucleic acids are described in detail in by Tijssen
ed., (1993): Chapter 3 of Laboratory Techniques in Biochemistry and
Molecular Biology: Hybridization With Nucleic Acid Probes, Part I.
Theory and Nucleic Acid Preparation, Elsevier, N.Y. and Tijssen
ed.
[0130] In a preferred embodiment, the "total" nucleic acid is
isolated from a given sample using, for example, an acid
guanidinium-phenol-chloroform extraction method and polyA+mRNA is
isolated by oligo dT column chromatography or by using (dT)n
magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, (1989), or Current Protocols in Molecular Biology, F.
Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New
York (1987)).
[0131] Frequently, it is desirable to amplify the nucleic acid
sample prior to assaying for gene copy number or expression level.
One of skill in the art will appreciate that whatever amplification
method is used, if a quantitative result is desired, care must be
taken to use a method that maintains or controls for the relative
frequencies of the amplified nucleic acids.
[0132] Methods of "quantitative" amplification are well known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction.
[0133] One preferred internal standard is a synthetic AW106 cRNA.
The AW106 cRNA is combined with RNA isolated from the sample
according to standard techniques known to those of skill in the
art. The RNA is then reverse transcribed using a reverse
transcriptase to provide copy DNA. The cDNA sequences are then
amplified (e.g., by PCR) using labeled primers. The amplification
products are separated, typically by electrophoresis, and the
amount of radioactivity (proportional to the amount of amplified
product) is determined. The amount of mRNA in the sample is then
calculated by comparison with the signal produced by the known
AW106 RNA standard. Detailed protocols for quantitative PCR are
provided in PCR Protocols, A Guide to Methods and Applications,
Innis et al., Academic Press, Inc. N.Y., (1990).
[0134] In a particularly preferred embodiment, where it is desired
to quantify the transcription level (and thereby expression) of a
one or more genes in a sample, the nucleic acid sample is one in
which the concentration of the mRNA transcript(s) of the gene or
genes, or the concentration of the nucleic acids derived from the
mRNA transcript(s), is proportional to the transcription level (and
therefore expression level) of that gene. Similarly, it is
preferred that the hybridization signal intensity be proportional
to the amount of hybridized nucleic acid. While it is preferred
that the proportionality be relatively strict (e.g., a doubling in
transcription rate results in a doubling in mRNA transcript in the
sample nucleic acid pool and a doubling in hybridization signal),
one of skill will appreciate that the proportionality can be more
relaxed and even non-linear. Thus, for example, an assay where a 5
fold difference in concentration of the target mRNA results in a 3
to 6 fold difference in hybridization intensity is sufficient for
most purposes. Where more precise quantification is required
appropriate controls can be run to correct for variations
introduced in sample preparation and hybridization as described
herein. In addition, serial dilutions of "standard" target nucleic
acids (e.g., mRNAs) can be used to prepare calibration curves
according to methods well known to those of skill in the art. Of
course, where simple detection of the presence or absence of a
transcript or large differences of changes in nucleic acid
concentration is desired, no elaborate control or calibration is
required.
[0135] In the simplest embodiment, such a nucleic acid sample is
the total mRNA or a total cDNA isolated and/or otherwise derived
from a biological sample. The nucleic acid (either genomic DNA or
mRNA) may be isolated from the sample according to any of a number
of methods well known to those of skill in the art as indicated
above.
[0136] 2) Hybridization-Based Assays.
[0137] i) Detection of Copy Number.
[0138] One method for evaluating the copy number of an MT-SP1 DNA
in a sample involves a Southern transfer. In a Southern Blot, the
DNA (e.g., genomic DNA), typically fragmented and separated on an
electrophoretic gel, is hybridized to a probe specific for the
target region. Comparison of the intensity of the hybridization
signal from the probe for the target region with control probe
signal from analysis of normal genomic DNA (e.g., a non-amplified
portion of the same or related cell, tissue, organ, etc.) provides
an estimate of the relative copy number of the target nucleic
acid.
[0139] An alternative means for determining the copy number of an
MT-SP1 gene of this invention is in situ hybridization. In situ
hybridization assays are well known (e.g., Angerer (1987) Meth.
Enzymol 152: 649). Generally, in situ hybridization comprises the
following major steps: (1) fixation of tissue or biological
structure to be analyzed; (2) prehybridization treatment of the
biological structure to increase accessibility of target DNA, and
to reduce nonspecific binding; (3) hybridization of the mixture of
nucleic acids to the nucleic acid in the biological structure or
tissue; (4) post-hybridization washes to remove nucleic acid
fragments not bound in the hybridization and (5) detection of the
hybridized nucleic acid fragments. The reagent used in each of
these steps and the conditions for use vary depending on the
particular application.
[0140] Preferred hybridization-based assays include, but are not
limited to, traditional "direct probe" methods such as Southern
blots or in situ hybridization (e.g., FISH), and "comparative
probe" methods such as comparative genomic hybridization (CGH). The
methods can be used in a wide variety of formats including, but not
limited to substrate- (e.g. membrane or glass) bound methods or
array-based approaches as described below.
[0141] In a typical in situ hybridization assay, cells are fixed to
a solid support, typically a glass slide. If a nucleic acid is to
be probed, the cells are typically denatured with heat or alkali.
The cells are then contacted with a hybridization solution at a
moderate temperature to permit annealing of Labeled probes specific
to the nucleic acid sequence encoding the protein. The targets
(e.g., cells) are then typically washed at a predetermined
stringency or at an increasing stringency until an appropriate
signal to noise ratio is obtained.
[0142] The probes are typically labeled, e.g., with radioisotopes
or fluorescent reporters as described above. Preferred probes are
sufficiently long so as to specifically hybridize with the target
nucleic acid(s) under stringent conditions. The preferred size
range is from about 20 bases to about 500 bases, more preferably
from about 30 bases to about 400 bases and most preferably from
about 40 bases to about 300 bases.
[0143] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. Thus, in some
embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block
non-specific hybridization.
[0144] Another effective approach for the quantification of copy
number of the gene(s) or EST(s) of this invention is comparative
genomic hybridization. In this method, a first collection of
(sample) nucleic acids (e.g. from a test sample derived from an
organism, tissue, or cell exposed to one or more drugs of abuse) is
labeled with a first label, while a second collection of (control)
nucleic acids (e.g. from a normal "unexposed" organism, tissue, or
cell) is labeled with a second label. The ratio of hybridization of
the nucleic acids is determined by the ratio of the two (first and
second) labels binding to each fiber in the array. Where there are
chromosomal deletions or multiplications, differences in the ratio
of the signals from the two labels will be detected and the ratio
will provide a measure of the gene and/or EST copy number.
[0145] Hybridization protocols suitable for use with the methods of
the invention are described, e.g., in Albertson (1984) EMBO J. 3:
1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142;
EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In
Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J.
(1994), etc. In one particularly preferred embodiment, the
hybridization protocol of Pinkel et al. (1998) Nature Genetics 20:
207-211, or of Kallioniemi (1992) Proc. Natl. Acad Sci USA
89:5321-5325 (1992) is used.
[0146] ii) Detection of Gene Transcript.
[0147] Methods of detecting and/or quantifying the transcript(s) of
one or more MT-SP1 gene(s) or EST(s) (e.g. mRNA or cDNA made
therefrom) using nucleic acid hybridization techniques are known to
those of skill in the art (see Sambrook et al. supra). For example,
one method for evaluating the presence, absence, or quantity of
gene or EST reverse-transcribed cDNA involves a Southern transfer
as described above. Alternatively, in a Northern blot, mRNA is
directly quantitated. In brief, the mRNA is isolated from a given
cell sample using, for example, an acid
guanidinium-phenol-chloroform extraction method. The mRNA is then
electrophoresed to separate the mRNA species and the mRNA is
transferred from the gel to a nitrocellulose membrane. As with the
Southern blots, labeled probes are used to identify and/or quantify
the target mRNA.
[0148] The probes used herein for detection of the MT-SP1 gene(s)
and/or EST(s) of this invention can be full length or less than the
full length of the gene or EST. Shorter probes are empirically
tested for specificity. Preferably nucleic acid probes are 20 bases
or longer in length. (see Sambrook et al. for methods of selecting
nucleic acid probe sequences for use in nucleic acid
hybridization.) Visualization of the hybridized portions allows the
qualitative determination of the presence or absence of gene(s)
and/or EST(s) of this invention.
[0149] 3) Amplification-Based Assays.
[0150] In still another embodiment, amplification-based assays can
be used to measure or level of gene (or EST) transcript. In such
amplification-based assays, the target nucleic acid sequences act
as template(s) in amplification reaction(s) (e.g. Polymerase Chain
Reaction (PCR) or reverse-transcription PCR (RT-PCR)). In a
quantitative amplification, the amount of amplification product
will be proportional to the amount of template in the original
sample. Comparison to appropriate (e.g. healthy tissue unexposed to
drug(s) of abuse) controls provides a measure of the copy number or
transcript level of the target gene or EST.
[0151] Methods of "quantitative" amplification are well known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided in Innis et al. (1990) PCR Protocols,
A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
The known nucleic acid sequence(s) for the cDNA, genes, and ESTs
provided herein is sufficient to enable one of skill to routinely
select primers to amplify any portion of the gene.
[0152] Other suitable amplification methods include, but are not
limited to ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and
Barringer et al. (1990) Gene 89: 117, transcription amplification
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173),
self-sustained sequence replication (Guatelli et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR,
etc.
[0153] As indicated above, PCR assay methods are well known to
those of skill in the art. Similarly, RT-PCR methods are also well
known.
[0154] 4) Hybridization Formats and Optimization of Hybridization
Conditions.
[0155] i) Array-Based Hybridization Formats.
[0156] In one embodiment, the methods of this invention can be
utilized in array-based hybridization formats. Arrays are a
multiplicity of different "probe" or "target" nucleic acids (or
other compounds) attached to one or more surfaces (e.g., solid,
membrane, or gel). In a preferred embodiment, the multiplicity of
nucleic acids (or other moieties) is attached to a single
contiguous surface or to a multiplicity of surfaces juxtaposed to
each other.
[0157] In an array format a large number of different hybridization
reactions can be run essentially "in parallel." This provides
rapid, essentially simultaneous, evaluation of a number of
hybridizations in a single "experiment". Methods of performing
hybridization reactions in array based formats are well known to
those of skill in the art (see, e.g., Pastinen (1997) Genome Res.
7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee
(1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature
Genetics 20: 207-211).
[0158] Arrays, particularly nucleic acid arrays can be produced
according to a wide variety of methods well known to those of skill
in the art. For example, in a simple embodiment, "low density"
arrays can simply be produced by spotting (e.g. by hand using a
pipette) different nucleic acids at different locations on a solid
support (e.g. a glass surface, a membrane, etc.).
[0159] This simple spotting, approach has been automated to produce
high density spotted arrays (see, e.g., U.S. Pat. No. 5,807,522).
This patent describes the use of an automated system that taps a
microcapillary against a surface to deposit a small volume of a
biological sample. The process is repeated to generate high density
arrays.
[0160] Arrays can also be produced using oligonucleotide synthesis
technology. Thus, for example, U.S. Pat. No. 5,143,854 and PCT
Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of
light-directed combinatorial synthesis of high density
oligonucleotide arrays. Synthesis of high density arrays is also
described in U.S. Pat. Nos. 5,744,305, 5,800,992 and 5,445,934.
[0161] In a preferred embodiment, the arrays used in this invention
comprise "probe" nucleic acids. These probes or target nucleic
acids are then hybridized respectively with their "target" nucleic
acids (e.g., mRNA derived from a biological sample).
[0162] In another embodiment the array, particularly a spotted
array, can include genomic DNA, e.g. one or more clones that
provide a high resolution scan of the genome containing the gene(s)
and/or EST(s) of this invention. The nucleic acid clones can be
obtained from, e.g., HACs, MACs, YACs, BACs, PACs, PIs, cosmids,
plasmids, inter-Alu PCR products of genomic clones, restriction
digests of genomic clones, cDNA clones, amplification (e.g., PCR)
products, and the like.
[0163] In various embodiments, the array nucleic acids are derived
from previously mapped libraries of clones spanning or including
the sequences of the invention. The arrays can be hybridized with a
single population of sample nucleic acid or can be used with two
differentially labeled collections (as with a test sample and a
reference sample).
[0164] Many methods for immobilizing nucleic acids on a variety of
solid surfaces are known in the art. A wide variety of organic and
inorganic polymers, as well as other materials, both natural and
synthetic, can be employed as the material for the solid surface.
Illustrative solid surfaces include, e.g., nitrocellulose, nylon,
glass, quartz, diazotized membranes (paper or nylon), silicones,
polyformaldehyde, cellulose, and cellulose acetate. In addition,
plastics such as polyethylene, polypropylene, polystyrene, and the
like can be used. Other materials which may be employed include
paper, ceramics, metals, metalloids, semiconductive materials,
cermets or the like. In addition, substances that form gels can be
used. Such materials include, e.g. proteins (e.g., gelatins),
lipopolysaccharides, silicates, agarose and polyacrylamides. Where
the solid surface is porous, various pore sizes may be employed
depending upon the nature of the system.
[0165] In preparing the surface, a plurality of different materials
may be employed, particularly as laminates, to obtain various
properties. For example, proteins (e.g., bovine serum albumin) or
mixtures of macromolecules (e.g., Denhardt's solution) can be
employed to avoid non-specific binding, simplify covalent
conjugation, enhance signal detection or the like. If covalent
bonding between a compound and the surface is desired, the surface
will usually be polyfunctional or be capable of being
polyfunctionalized. Functional groups which may be present on the
surface and used for linking can include carboxylic acids,
aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl
groups, mercapto groups and the like. The manner of linking a wide
variety of compounds to various surfaces is well known and is amply
illustrated in the literature.
[0166] For example, methods for immobilizing nucleic acids by
introduction of various functional groups to the molecules is known
(see, e.g., Bischoff (1987) Anal. Biochem., 164: 336-344; Kremsky
(1987) Nucl. Acids Res. 15: 2891-2910). Modified nucleotides can be
placed on the target using PCR primers containing the modified
nucleotide, or by enzymatic end labeling with modified nucleotides.
Use of glass or membrane supports (e.g., nitrocellulose, nylon,
polypropylene) for the nucleic acid arrays of the invention is
advantageous because of well developed technology employing manual
and robotic methods of arraying targets at relatively high element
densities. Such membranes are generally available and protocols and
equipment for hybridization to membranes is well known.
[0167] Target elements of various sizes, ranging from 1 mm diameter
down to 1 .mu.m can be used. Relatively simple approaches capable
of quantitative fluorescent imaging of 1 cm.sup.2 areas have been
described that permit acquisition of data from a large number of
target elements in a single image (see, e.g., Wittrup (1994)
Cytometry 16:206-213, Pinkel et al. (1998) Nature Genetics 20:
207-211).
[0168] Arrays on solid surface substrates with much lower
fluorescence than membranes, such as glass, quartz, or small beads,
can achieve much better sensitivity. Substrates such as glass or
fused silica are advantageous in that they provide a very low
fluorescence substrate, and a highly efficient hybridization
environment. Covalent attachment of the target nucleic acids to
glass or synthetic fused silica can be accomplished according to a
number of known techniques (described above). Nucleic acids can be
conveniently coupled to glass using commercially available
reagents. For instance, materials for preparation of silanized
glass with a number of functional groups are commercially available
or can be prepared using standard techniques (see, e.g., Gait
(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press,
Wash., D.C.). Quartz cover slips, which have at least 10-fold lower
autofluorescence than glass, can also be silanized.
[0169] Alternatively, probes can also be immobilized on
commercially available coated beads or other surfaces. For
instance, biotin end-labeled nucleic acids can be bound to
commercially available avidin-coated beads. Streptavidin or
anti-digoxigenin antibody can also be attached to silanized glass
slides by protein-mediated coupling using e.g., protein A following
standard protocols (see, e.g., Smith (1992) Science 258:
1122-1126). Biotin or digoxigenin end-labeled nucleic acids can be
prepared according to standard techniques. Hybridization to nucleic
acids attached to beads is accomplished by suspending them in the
hybridization mix, and then depositing them on the glass substrate
for analysis after washing. Alternatively, paramagnetic particles,
such as ferric oxide particles, with or without avidin coating, can
be used.
[0170] ii) Other Hybridization Formats.
[0171] A variety of nucleic acid hybridization formats are known to
those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in Harnes and
Higgins (1985) Nucleic Acid Hybridization, A Practical Approach,
IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63:
378-383; and John et al. (1969) Nature 223: 582-587.
[0172] Sandwich assays are commercially useful hybridization assays
for detecting or isolating nucleic acid sequences. Such assays
utilize a "capture" nucleic acid covalently immobilized to a solid
support and a labeled "signal" nucleic acid in solution. The sample
will provide the target nucleic acid. The "capture" nucleic acid
and "signal" nucleic acid probe hybridize with the target nucleic
acid to form a "sandwich" hybridization complex. To be most
effective, the signal nucleic acid should not hybridize with the
capture nucleic acid.
[0173] Typically, labeled signal nucleic acids are used to detect
hybridization. Complementary nucleic acids or signal nucleic acids
may be labeled by any one of several methods typically used to
detect the presence of hybridized polynucleotides. The most common
method of detection is the use of autoradiography with .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P-labelled probes or the
like. Other labels include ligands that bind to labeled antibodies,
fluorophores, chemi-luminescent agents, enzymes, and antibodies
which can serve as specific binding pair members for a labeled
ligand.
[0174] Detection of a hybridization complex may require the binding
of a signal generating complex to a duplex of target and probe
polynucleotides or nucleic acids. Typically, such binding occurs
through ligand and anti-ligand interactions as between a
ligand-conjugated probe and an anti-ligand conjugated with a
signal.
[0175] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system that multiplies
the target nucleic acid being detected. Examples of such systems
include the polymerase chain reaction (PCR) system and the ligase
chain reaction (LCR) system. Other methods recently described in
the art are the nucleic acid sequence based amplification (NASBAO,
Cangene, Mississauga, Ontario) and Q Beta Replicase systems.
[0176] iii) Optimization of Hybridization Conditions.
[0177] Nucleic acid hybridization simply involves providing a
denatured probe and target nucleic acid under conditions where the
probe and its complementary target can form stable hybrid duplexes
through complementary base pairing. The nucleic acids that do not
form hybrid duplexes are then washed away leaving the hybridized
nucleic acids to be detected, typically through detection of an
attached detectable label. It is generally recognized that nucleic
acids are denatured by increasing the temperature or decreasing the
salt concentration of the buffer containing the nucleic acids, or
in the addition of chemical agents, or the raising of the pH. Under
low stringency conditions (e.g., low temperature and/or high salt
and/or high target concentration) hybrid duplexes (e.g., DNA:DNA,
RNA:RNA, or RNA:DNA) will form even where the annealed sequences
are not perfectly complementary. Thus specificity of hybridization
is reduced at lower stringency. Conversely, at higher stringency
(e.g., higher temperature or lower salt) successful hybridization
requires fewer mismatches.
[0178] One of skill in the art will appreciate that hybridization
conditions may be selected to provide any degree of stringency. In
a preferred embodiment, hybridization is performed at low
stringency to ensure hybridization and then subsequent washes are
performed at higher stringency to eliminate mismatched hybrid
duplexes. Successive washes may be performed at increasingly higher
stringency (e.g., down to as low as 0.25.times.SSPE at 37.degree.
C. to 70.degree. C.) until a desired level of hybridization
specificity is obtained. Stringency can also be increased by
addition of agents such as formamide. Hybridization specificity may
be evaluated by comparison of hybridization to the test probes with
hybridization to the various controls that can be present.
[0179] In general, there is a tradeoff between hybridization
specificity (stringency) and signal intensity. Thus, in a preferred
embodiment, the wash is performed at the highest stringency that
produces consistent results and that provides a signal intensity
greater than approximately 10% of the background intensity. Thus,
in a preferred embodiment, the hybridized array may be washed at
successively higher stringency solutions and read between each
wash. Analysis of the data sets thus produced will reveal a wash
stringency above which the hybridization pattern is not appreciably
altered and which provides adequate signal for the particular
probes of interest.
[0180] In a preferred embodiment, background signal is reduced by
the use of a blocking reagent (e.g., tRNA, sperm DNA, cot-1 DNA,
etc.) during the hybridization to reduce non-specific binding. The
use of blocking agents in hybridization is well known to those of
skill in the art (see, e.g., Chapter 8 in P. Tijssen, supra.)
[0181] Methods of optimizing hybridization conditions are well
known to those of skill in the art (see, e.g., Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular Biology, Vol
24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.).
[0182] Optimal conditions are also a function of the sensitivity of
label (e.g., fluorescence) detection for different combinations of
substrate type, fluorochrome, excitation and emission bands, spot
size and the like. Low fluorescence background surfaces can be used
(see, e.g., Chu (1992) Electrophoresis 13:105-114). The sensitivity
for detection of spots ("target elements") of various diameters on
the candidate surfaces can be readily determined by, e.g., spotting
a dilution series of fluorescently end labeled DNA fragments. These
spots are then imaged using conventional fluorescence microscopy.
The sensitivity, linearity, and dynamic range achievable from the
various combinations of fluorochrome and solid surfaces (e.g.,
glass, fused silica, etc.) can thus be determined. Serial dilutions
of pairs of fluorochrome in known relative proportions can also be
analyzed. This determines the accuracy with which fluorescence
ratio measurements reflect actual fluorochrome ratios over the
dynamic range permitted by the detectors and fluorescence of the
substrate upon which the probe has been fixed.
[0183] iv) Labeling and Detection of Nucleic Acids.
[0184] In a preferred embodiment, the hybridized nucleic acids are
detected by detecting one or more labels attached to the sample
nucleic acids. The labels may be incorporated by any of a number of
means well known to those of skill in the art. Means of attaching
labels to nucleic acids include, for example nick translation, or
end-labeling by kinasing of the nucleic acid and subsequent
attachment (ligation) of a linker joining the sample nucleic acid
to a label (e.g., a fluorophore). A wide variety of linkers for the
attachment of labels to nucleic acids are also known. In addition,
intercalating dyes and fluorescent nucleotides can also be
used.
[0185] Detectable labels suitable for use in the present invention
include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include biotin for staining
with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like, see, e.g.,
Molecular Probes, Eugene, Oreg., USA), radiolabels (e.g., .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse
radish peroxidase, alkalinephosphatase and others commonly used in
an ELISA), and calorimetric labels such as colloidal gold (e.g.,
gold particles in the 40-80 nm diameter size range scatter green
light with high efficiency) or colored glass or plastic (e.g.,
polystyrene, polypropylene, latex, etc.) beads. Patents teaching
the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
[0186] A fluorescent label is preferred because it provides a very
strong signal with low background. It is also optically detectable
at high resolution and sensitivity through a quick scanning
procedure. The nucleic acid samples can all be labeled with a
single label, e.g., a single fluorescent label. Alternatively, in
another embodiment, different nucleic acid samples can be
simultaneously hybridized where each nucleic acid sample has a
different label. For instance, one target could have a green
fluorescent label and a second target could have a red fluorescent
label. The scanning step will distinguish sites of binding of the
red label from those binding the green fluorescent label. Each
nucleic acid sample (target nucleic acid) can be analyzed
independently from one another.
[0187] Suitable chromogens which can be employed include those
molecules and compounds which absorb light in a distinctive range
of wavelengths so that a color can be observed or, alternatively,
which emit light when irradiated with radiation of a particular
wave length or wave length range, e.g., fluorescers.
[0188] Desirably, fluorescers should absorb light above about 300
nm, preferably about 350 nm, and more preferably above about 400
nm, usually emitting at wavelengths greater than about 10 nm higher
than the wavelength of the light absorbed. It should be noted that
the absorption and emission characteristics of the bound dye can
differ from the unbound dye. Therefore, when referring to the
various wavelength ranges and characteristics of the dyes, it is
intended to indicate the dyes as employed and not the dye which is
unconjugated and characterized in an arbitrary solvent.
[0189] Fluorescers are generally preferred because by irradiating a
fluorescer with light, one can obtain a plurality of emissions.
Thus, a single label can provide for a plurality of measurable
events.
[0190] Detectable signal can also be provided by chemiluminescent
and bioluminescent sources. Chemiluminescent sources include a
compound which becomes electronically excited by a chemical
reaction and can then emit light which serves as the detectable
signal or donates energy to a fluorescent acceptor. Alternatively,
luciferins can be used in conjunction with luciferase or lucigenins
to provide bioluminescence.
[0191] Spin labels are provided by reporter molecules with an
unpaired electron spin which can be detected by electron spin
resonance (ESR) spectroscopy. Exemplary spin labels include organic
free radicals, transitional metal complexes, particularly vanadium,
copper, iron, and manganese, and the like. Exemplary spin labels
include nitroxide free radicals.
[0192] The label may be added to the target (sample) nucleic
acid(s) prior to, or after the hybridization. So called "direct
labels" are detectable labels that are directly attached to or
incorporated into the target (sample) nucleic acid prior to
hybridization. In contrast, so called "indirect labels" are joined
to the hybrid duplex after hybridization. Often, the indirect label
is attached to a binding moiety that has been attached to the
target nucleic acid prior to the hybridization. Thus, for example,
the target nucleic acid may be biotinylated before the
hybridization. After hybridization, an avidin-conjugated
fluorophore will bind the biotin bearing hybrid duplexes providing
a label that is easily detected. For a detailed review of methods
of labeling nucleic acids and detecting labeled hybridized nucleic
acids see Laboratory Techniques in Biochemistry and Molecular
Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P.
Tijssen, ed. Elsevier, N.Y., (1993)).
[0193] Fluorescent labels are easily added during an in vitro
transcription reaction. Thus, for example, fluorescein labeled UTP
and CTP can be incorporated into the RNA produced in an in vitro
transcription.
[0194] The labels can be attached directly or through a linker
moiety. In general, the site of label or linker-label attachment is
not limited to any specific position. For example, a label may be
attached to a nucleoside, nucleotide, or analogue thereof at any
position that does not interfere with detection or hybridization as
desired. For example, certain Label-ON Reagents from Clontech (Palo
Alto, Calif.) provide for labeling interspersed throughout the
phosphate backbone of an oligonucleotide and for terminal labeling
at the 3' and 5' ends. As shown for example herein, labels can be
attached at positions on the ribose ring or the ribose can be
modified and even eliminated as desired. The base moieties of
useful labeling reagents can include those that are naturally
occurring or modified in a manner that does not interfere with the
purpose to which they are put. Modified bases include but are not
limited to 7-deaza A and G, 7-deaza-8-aza A and G, and other
heterocyclic moieties.
[0195] It will be recognized that fluorescent labels are not to be
limited to single species organic molecules, but include inorganic
molecules, multi-molecular mixtures of organic and/or inorganic
molecules, crystals, heteropolymers, and the like. Thus, for
example, CdSe-CdS core-shell nanocrystals enclosed in a silica
shell can be easily derivatized for coupling to a biological
molecule (Bruchez et al. (1998) Science, 281: 2013-2016).
Similarly, highly fluorescent quantum dots (zinc sulfide-capped
cadmium selenide) have been covalently coupled to biomolecules for
use in ultrasensitive biological detection (Warren and Nie (1998)
Science, 281: 2016-2018).
[0196] C) Polypeptide-Based Assays.
[0197] In addition to, or in alternative to, the detection of
nucleic acid level(s), alterations in expression of MT-SP1 can be
detected and/or quantified by detecting and/or quantifying the
amount and/or activity of translated MT-SP1 protein(s). In
particularly preferred embodiments, the MT-SP1 proteins are
detected immunohistochemically, using a radioimmunoassay, or using
other immunoassay(s). As used herein, an immunoassay is an assay
that utilizes an antibody to specifically bind to the analyte
(MT-SP1). The immunoassay is thus characterized by detection of
specific binding of a MT-SP1 protein., or protein fragment, to an
anti-MT-SP1 antibody as opposed to the use of other physical or
chemical properties to isolate, target, and quantify the
analyte.
[0198] 1) Detection of Expressed Protein
[0199] The MT-SP1 polypeptide(s) of this invention can be detected
and quantified by any of a number of methods well known to those of
skill in the art. These may include analytic biochemical methods
such as electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, or various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunohistochemistry, affinity
chromatography, immunoelectrophoresis, radioimmunoassay (RIA),
enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
assays, western blotting, and the like.
[0200] In one preferred embodiment, the MT-SP1 polypeptide(s) are
detected and/or quantified using immunohistochemical methods. In
this approach, antibodies that specifically bind to an MT-SP1 are
contacted with the biological sample (e.g., a histological sample).
Those antibodies that specifically bind to the sample are
visualized, or otherwise detected, and provide an indication of the
location, presence, absence or quantity of MT-SP1 protein in the
sample. The antibodies are typically detected by detection of a
label either affixed to the antibody prior to or subsequent to the
"contacting" step. Immunohistochemical methods are well known to
those of skill in the art (see, e.g., Kleihues et al. (1993),
Histological typing of tumours of the central nervous system,
Springer Verlag, New York).
[0201] In another preferred embodiment, the MT-SP1 polypeptide(s)
are detected/quantified in an electrophoretic protein separation
(e.g. a 1- or 2-dimensional electrophoresis). Means of detecting
proteins using electrophoretic techniques are well known to those
of skill in the art (see generally, R. Scopes (1982) Protein
Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods in
Enzymology Vol. 182: Guide to Protein Purification, Academic Press,
Inc., N.Y.).
[0202] Another preferred embodiment utilizes a Western blot
(immunoblot) analysis to detect and quantify the presence of
polypeptide(s) of this invention in the sample. This technique
generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with the antibodies that
specifically bind the target polypeptide(s).
[0203] The antibodies specifically bind to the target
polypeptide(s) and may be directly labeled or alternatively may be
subsequently detected using labeled antibodies (e.g., labeled sheep
anti-mouse antibodies) that specifically bind to the a domain of
the antibody.
[0204] Other suitable assay formats include, but are not limited
to, liposome immunoassays (LIA), which use liposomes designed to
bind specific molecules (e.g., antibodies) and release encapsulated
reagents or markers. The released chemicals are then detected
according to standard techniques (see, Monroe et al. (1986) Amer.
Clin. Prod. Rev. 5: 34-41).
[0205] In a preferred embodiment, the MT-SP1 protein(s) are
detected and/or quantified in the biological sample using any of a
number of well recognized immunological binding assays
(immunoassays) (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Methods in Cell Biology Volume 37:
Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York
(1993); Basic and Clinical Immunology 7th Edition, Stites &
Terr, eds. (1991).
[0206] As used herein, an immunoassay is an assay that utilizes an
antibody to specifically bind to the analyte (e.g., an MT-SP1
polypeptide). The immunoassay is thus characterized by detection of
specific binding of a polypeptide of this invention to an antibody
as opposed to the use of other physical or chemical properties to
isolate, target, and quantify the analyte.
[0207] Immunological binding assays (or immunoassays) typically
utilize a "capture agent" to specifically bind to and often
immobilize the analyte (in this case an MT-SP1 polypeptide). In
preferred embodiments, the capture agent is an antibody.
[0208] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled polypeptide or a labeled antibody
that specifically recognizes the already bound target polypeptide.
Alternatively, the labeling agent may be a third moiety, such as
another antibody, that specifically binds to the capture
agent/polypeptide complex.
[0209] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G may
also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally Kronval,
et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J.
Immunol., 135: 2589-2542).
[0210] As indicated above, immunoassays for the detection and/or
quantification of the MT-SP1 polypeptide(s) of this invention can
take a wide variety of formats well known to those of skill in the
art. Preferred immunoassays for detecting the target polypeptide(s)
are either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of captured analyte is
directly measured. In one preferred "sandwich" assay, for example,
the capture agents (antibodies) can be bound directly to a solid
substrate where they are immobilized. These immobilized antibodies
then capture the target polypeptide present in the test sample. The
target polypeptide thus immobilized is then bound by a labeling
agent, such as a second antibody bearing a label.
[0211] In competitive assays, the amount of analyte (MT-SP1
polypeptide) present in the sample is measured indirectly by
measuring the amount of an added (exogenous) analyte displaced (or
competed away) from a capture agent (antibody) by the analyte
present in the sample. In one competitive assay, a known amount of,
in this case, labeled polypeptide is added to the sample and the
sample is then contacted with a capture agent. The amount of
labeled polypeptide bound to the antibody is inversely proportional
to the concentration of target polypeptide present in the
sample.
[0212] In one particularly preferred embodiment, the antibody is
immobilized on a solid substrate. The immobilized antibody captures
the target MT-SP1 thereby immobilizing the analyte. The amount of
analyte (target polypeptide) bound to the antibody may be
determined either by measuring the amount of target polypeptide
present in the polypeptide/antibody complex, or alternatively by
measuring the amount of remaining uncomplexed polypeptide.
[0213] The assays of this invention are scored (as positive or
negative or quantity of target polypeptide) according to standard
methods well known to those of skill in the art. The particular
method of scoring will depend on the assay format and choice of
label. For example, a Western Blot assay can be scored by
visualizing the colored product produced by the enzymatic label. A
clearly visible colored band or spot at the correct molecular
weight is scored as a positive result, while the absence of a
clearly visible spot or band is scored as a negative. The intensity
of the band or spot can provide a quantitative measure of target
polypeptide concentration.
[0214] Antibodies for use in the various immunoassays described
herein, can be produced as described below.
[0215] 2) Detection of Enzyme Activity.
[0216] In another embodiment, levels of gene expression/regulation
are assayed by measuring the enzymatic activity of the polypeptide
encoded by the respective gene(s). For example, the MT-SP1
polypeptide(s) of this invention are serine proteases and their
activity can be readily detected by assaying the cleavage of a
target substrate. Thus, Example 1 illustrated quantification of
MT-SP1 activity using an active site titration with MUGB. The
catalytic activity of the protease domain can also be monitored
using pNA substrates. In particular, MT-SP1 protease activity can
be tested against tetrapeptide substrates of the form Suc-AAPX-pNA,
which contained various amino acids at the P1 position (P1-Ala,
Asp, Glu, Phe, Leu, Met, Lys, or Arg). In a preferred embodiment,
substrates with P1-Lys or P1-Arg are used. Protease domain can also
be characterized using the substrate Spectrozyme tPA
(hexahydrotyrosyl-Gly-Arg-pNA) as described in Example 1. Using the
teaching provided herein, assays for activity of other MT-SP1
proteases are easily performed.
[0217] 3) Antibodies to Polypeptides Expressed by the Genes or ESTs
Identified Herein.
[0218] Either polyclonal or monoclonal antibodies may be used in
the immunoassays of the invention described herein. Polyclonal
antibodies are preferably raised by multiple injections (e.g.
subcutaneous or intramuscular injections) of substantially pure
polypeptides or antigenic polypeptides into a suitable non-human
mammal. The antigenicity of the target peptides can be determined
by conventional techniques to determine the magnitude of the
antibody response of an animal that has been immunized with the
peptide. Generally, the peptides that are used to raise antibodies
for use in the methods of this invention should generally be those
which induce production of high titers of antibody with relatively
high affinity for target polypeptides encoded by the MT-SP1 genes
or ESTs of this invention.
[0219] If desired, the immunizing peptide may be coupled to a
carrier protein by conjugation using techniques that are well-known
in the art. Such commonly used carriers which are chemically
coupled to the peptide include keyhole limpet hemocyanin (KLH),
thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The
coupled peptide is then used to immunize the animal (e.g. a mouse
or a rabbit).
[0220] The antibodies are then obtained from blood samples taken
from the mammal The techniques used to develop polyclonal
antibodies are known in the art (see, e.g., Methods of Enzymology,
"Production of Antisera With Small Doses of Immunogen: Multiple
Intradermal Injections", Langone, et al. eds. (Acad. Press, 1981)).
Polyclonal antibodies produced by the animals can be further
purified, for example, by binding to and elution from a matrix to
which the peptide to which the antibodies were raised is bound.
Those of skill in the art will know of various techniques common in
the immunology arts for purification and/or concentration of
polyclonal antibodies, as well as monoclonal antibodies sees for
example, Coligan, et al. (1991) Unit 9, Current Protocols in
Immunology, Wiley Interscience).
[0221] Preferably, however, the antibodies produced will be
monoclonal antibodies ("mAb's"). For preparation of monoclonal
antibodies, immunization of a mouse or rat is preferred. The term
"antibody" as used in this invention includes intact molecules as
well as fragments thereof, such as, Fab and F(ab').sup.2, and/or
single-chain antibodies (e.g. scFv) which are capable of binding an
epitopic determinant. Also, in this context, the term "mab's of the
invention" refers to monoclonal antibodies with specificity for a
polypeptide encoded by an MT-SP1 gene or EST.
[0222] The general method used for production of hybridomas
secreting mAbs is well known (Kohler and Milstein (1975) Nature,
256:495). Briefly, as described by Kohler and Milstein the
technique comprised isolating lymphocytes from regional draining
lymph nodes of five separate cancer patients with either melanoma,
teratocarcinoma or cancer of the cervix, glioma or lung, (where
samples were obtained from surgical specimens), pooling the cells,
and fusing the cells with SHFP-1. Hybridomas were screened for
production of antibody which bound to cancer cell lines.
Confirmation of specificity among mAb's can be accomplished using
relatively routine screening techniques (such as the enzyme-linked
immunosorbent assay, or "ELISA") to determine the elementary
reaction pattern of the mAb of interest.
[0223] It is also possible to evaluate an mAb to determine whether
it has the same specificity as a mAb of the invention without undue
experimentation by determining whether the mAb being tested
prevents a mAb of the invention from binding to the target
polypeptide isolated as described above. If the mAb being tested
competes with the mAb of the invention, as shown by a decrease in
binding by the mAb of the invention, then it is likely that the two
monoclonal antibodies bind to the same or a closely related
epitope. Still another way to determine whether a mAb has the
specificity of a mAb of the invention is to preincubate the mAb of
the invention with an antigen with which it is normally reactive,
and determine if the mAb being tested is inhibited in its ability
to bind the antigen. If the mAb being tested is inhibited then, in
all likelihood, it has the same, or a closely related, epitopic
specificity as the mAb of the invention.
[0224] Antibodies fragments, e.g. single chain antibodies (scFv or
others), can also be produced/selected using phage display
technology. The ability to express antibody fragments on the
surface of viruses that infect bacteria (bacteriophage or phage)
makes it possible to isolate a single binding antibody fragment,
e.g., from a library of greater than 10.sup.10 nonbinding clones.
To express antibody fragments on the surface of phage (phage
display), an antibody fragment gene is inserted into the gene
encoding a phage surface protein (e.g., pIII) and the antibody
fragment-pIII fusion protein is displayed on the phage surface
(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al.
(1991) Nucleic Acids Res. 19: 4133-4137).
[0225] Since the antibody fragments on the surface of the phage are
functional, phage bearing antigen binding antibody fragments can be
separated from non-binding phage by antigen affinity chromatography
(McCafferty et al. (1990) Nature, 348: 552-554). Depending on the
affinity of the antibody fragment, enrichment factors of 20 fold
-1,000,000 fold are obtained for a single round of affinity
selection. By infecting bacteria with the eluted phage, however,
more phage can be grown and subjected to another round of
selection. In this way, an enrichment of 1000 fold in one round can
become 1,000,000 fold in two rounds of selection (McCafferty et al.
(1990) Nature, 348: 552-554). Thus even when enrichments are low
(Marks et al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds
of affinity selection can lead to the isolation of rare phage.
Since selection of the phage antibody library on antigen results in
enrichment, the majority of clones bind antigen after as few as
three to four rounds of selection. Thus only a relatively small
number of clones (several hundred) need to be analyzed for binding
to antigen.
[0226] Human antibodies can be produced without prior immunization
by displaying very large and diverse V-gene repertoires on phage
Marks et al. (1991) J. Mol. Biol. 222: 581-597). In one embodiment
natural V.sub.H and V.sub.L repertoires present in human peripheral
blood lymphocytes are were isolated from unimmunized donors by PCR.
The V-gene repertoires were spliced together at random using PCR to
create a scFv gene repertoire which is was cloned into a phage
vector to create a library of 30 million phage antibodies (Id.).
From this single "naive" phage antibody library, binding antibody
fragments have been isolated against more than 17 different
antigens, including haptens, polysaccharides and proteins (Marks et
al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).
Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:
725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies
have been produced against self proteins, including human
thyroglobulin, immunoglobulin, tumor necrosis factor and CEA
(Griffiths et al. (1993) EMBO J. 12: 725-734). It is also possible
to isolate antibodies against cell surface antigens by selecting
directly on intact cells. The antibody fragments are highly
specific for the antigen used for selection and have affinities in
the 1 .mu.M to 100 nM range (Marks et al. (1991) J. Mol. Biol.
222:581-597; Griffiths et al. (1993) EMBO J. 12: 725-734). Larger
phage antibody libraries result in the isolation of more antibodies
of higher binding affinity to a greater proportion of antigens.
[0227] It will also be recognized that antibodies can be prepared
by any of a number of commercial services (e.g., Berkeley antibody
laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).
V. MT-SP1 as a Target for Screening for Therapeutics.
[0228] A) Screening Target for Agents that Modulate MT-SP1
Expression and/or Activity.
[0229] While, in one embodiment, the assays described above
provided methods of detecting the presence or absence, or
quantifying expression of an MT-SP1 protease, it will be
appreciated that the same assays can be used to screen for agents
that modulate the expression of and/or the activity of an MT-SP1
serine protease. To screen for potential modulators, the assays
described above are performed in the presence of one or more test
agents or are performed using biological samples from cells and/or
tissues and/or organs and/or organisms exposed to one or more test
agents. The MT-SP1 activity and/or expression level is determined
and, in a preferred embodiment, compared to the activity level(s)
observed in "control" assays (e.g., the same assays lacking the
test agent). A difference between the MT-SP1 expression and/or
activity in the "test" assay as compared to the control assay
indicates that the test agent is a "modulator" of SP1 expression
and/or activity.
[0230] In a preferred embodiment, the assays of this invention
level are deemed to show a positive result, e.g. elevated
expression and/or MT-SP1 activity, genes, when the measured protein
or nucleic acid level or protein activity is greater than the level
measured or known for a control sample (e.g. either a level known
or measured for a normal healthy cell, tissue or organism mammal of
the same species not exposed to the or putative modulator (test
agent), or a "baseline/reference" level determined at a different
tissue and/or a different time for the same individual). In a
particularly preferred embodiment, the assay is deemed to show a
positive result when the difference between sample and "control" is
statistically significant (e.g. at the 85% or greater, preferably
at the 90% or greater, more preferably at the 95% or greater and
most preferably at the 98% or greater confidence level).
[0231] B) Pre-Screening for Agents that Specifically Bind/Interact
with MT-SP1.
[0232] In certain embodiments it is desired to pre-screen test
agents for the ability to interact with (e.g. specifically bind to)
a MT-SP1 nucleic acid or polypeptide. Specifically binding test
agents are more likely to interact with and thereby modulate MT-SP1
expression and/or activity. Thus, in some preferred embodiments,
the test agent(s) are pre-screened for binding to MT-SP1 or to an
MT-SP1 nucleic acid before performing the more complex assays
described above.
[0233] In one embodiment, such pre-screening is accomplished with
simple binding assays. Means of assaying for specific binding or
the binding affinity of a particular ligand for a nucleic acid or
for a protein are well known to those of skill in the art. In
preferred binding assays, the MT-SP1 protein or nucleic acid is
immobilized and exposed to a test agent (which can be labeled), or
alternatively, the test agent(s) are immobilized and exposed to an
MT-SP1 or to a MT-SP1 nucleic acid (which can be labeled). The
immobilized moiety is then washed to remove any unbound material
and the bound test agent or bound MT-SP1 protein or nucleic acid is
detected (e.g. by detection of a label attached to the bound
molecule). The amount of immobilized label is proportional to the
degree of binding between the MT-SP1 protein or nucleic acid and
the test agent.
[0234] C) High Throughput Screening for MT-SP1 Modulators (e.g.,
Therapeutics).
[0235] The assays for modulators of MT-SP1 expression and/or
activity described herein are also amenable to "high-throughput"
modalities. Conventionally, new chemical entities with useful
properties (e.g., modulation of MT-SP1 activity or expression) are
generated by identifying a chemical compound (called a "lead
compound") with some desirable property or activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. However, the current trend is
to shorten the time scale for all aspects of drug discovery.
Because of the ability to test large numbers quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead compound identification methods.
[0236] In one preferred embodiment, high throughput screening
methods involve providing a library containing a large number of
compounds (candidate compounds) potentially having the desired
activity. Such "combinatorial chemical libraries" are then screened
in one or more assays, as described herein, to identify those
library members (particular chemical species or subclasses) that
display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0237] 1) Combinatorial Chemical Libraries for Potential Modulators
of MT-SP1.
[0238] The likelihood of an assay identifying a MT-SP1 expression
or activity modulator is increased when the number and types of
test agents used in the screening system is increased. Recently,
attention has focused on the use of combinatorial chemical
libraries to assist in the generation of new chemical compound
leads. A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks called amino acids in
every possible way for a given compound length (i.e., the number of
amino acids in a polypeptide compound). Millions of chemical
compounds can be synthesized through such combinatorial mixing of
chemical building blocks. For example, one commentator has observed
that the systematic, combinatorial mixing of 100 interchangeable
chemical building blocks results in the theoretical synthesis of
100 million tetrameric compounds or 10 billion pentameric compounds
(Gallop et al. (1994) 37(9): 1233-1250).
[0239] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991)
Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991)
Nature, 354: 84-88). Peptide synthesis is by no means the only
approach envisioned and intended for use with the present
invention. Other chemistries for generating chemical diversity
libraries can also be used. Such chemistries include, but are not
limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct.
1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993)
Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides
(Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et
al., (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic
syntheses of small compound libraries (Chen et al. (1994) J. Amer.
Chem. Soc. 116: 2661), oligocarbamates (Cho, et al., (1993) Science
261:1303), and/or peptidyl phosphonates (Campbell et al., (1994) J.
Org. Chem. 59: 658). See, generally, Gordon et al., (1994) J. Med.
Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene,
Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No.
5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996)
Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al. (1996) Science,
274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organic
molecule libraries (see, e.g., benzodiazepines, Baum (1993)
C&EN, Jan 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,
thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,
pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholino
compounds U.S. Pat. No. 5,506,337, benzodiazepines 5,288,514, and
the like).
[0240] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.).
[0241] A number of well known robotic systems have also been
developed for solution phase chemistries. These systems include
automated workstations like the automated synthesis apparatus
developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and
many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto,
Calif.) which mimic the manual synthetic operations performed by a
chemist. Any of the above devices are suitable for use with the
present invention. The nature and implementation of modifications
to these devices (if any) so that they can operate as discussed
herein will be apparent to persons skilled in the relevant art. In
addition, numerous combinatorial libraries are themselves
commercially available (see, e.g., ComGenex, Princeton, N.J.,
Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd,
Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences,
Columbia, Md., etc.).
[0242] 2) High Throughput Assays of Chemical Libraries for
Modulators of MT-SP1.
[0243] Any of the assays for agents that modulate MT-SP1 expression
and/or activity (e.g. that have potential therapeutic activity) are
amenable to high throughput screening. As described-above, having
identified the nucleic acid whose expression is altered upon
exposure to a drug of abuse, likely modulators either inhibit
expression of the gene product, or inhibit the activity of the
expressed protein. Preferred assays thus detect inhibition of
transcription (i.e., inhibition of mRNA production) by the test
compound(s), inhibition of protein expression by the test
compound(s), or binding to the gene (e.g., gDNA, or cDNA) or gene
product (e.g., mRNA or expressed protein) by the test compound(s).
Alternatively, the assay can detect inhibition of the
characteristic protease activity of the MT-SP1 gene product. High
throughput assays for the presence, absence, or quantification of
particular nucleic acids or protein products are well known to
those of skill in the art. Similarly, binding assays are similarly
well known. Thus, for example, U.S. Pat. No. 5,559,410 discloses
high throughput screening methods for proteins, U.S. Pat. No.
5,585,639 discloses high throughput screening methods for nucleic
acid binding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220 and
5,541,061 disclose high throughput methods of screening for
ligand/antibody binding.
[0244] In addition, high throughput screening systems are
commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.;
Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.
Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.).
These systems typically automate entire procedures including all
sample and reagent pipetting, liquid dispensing, timed incubations,
and final readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and
rapid start up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed
protocols the various high throughput. Thus, for example, Zymark
Corp. provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like.
VI. Assay Optimization.
[0245] The assays of this invention have immediate utility in
detecting elevated expression and/or activity of an MT-SP1 protease
or for screening for agents that modulate the MT-SP1 activity of a
cell, tissue or organism. The assays of this invention can be
optimized for use in particular contexts, depending, for example,
on the source and/or nature of the biological sample and/or the
particular test agents, and/or the analytic facilities
available.
[0246] Thus, for example, optimization can involve determining
optimal conditions for binding assays, optimum sample processing
conditions (e.g. preferred PCR conditions), hybridization
conditions that maximize signal to noise, protocols that improve
throughput, etc. In addition, assay formats can be selected and/or
optimized according to the availability of equipment and/or
reagents. Thus, for example, where commercial antibodies or ELISA
kits are available it may be desired to assay protein
concentration. Conversely, where it is desired to screen for
modulators that alter transcription of one or more of the genes or
ESTs identified herein, nucleic acid based assays are
preferred.
[0247] Routine selection and optimization of assay formats is well
known to those of ordinary skill in the art.
VII. MT-SP1-Targeted Therapeutics.
[0248] Since MT-SP1 is found in a cell membrane, it can be
exploited as target for the efficient and specific delivery of an
effector (e.g. an effector molecule such as a cytotoxin, a
radiolabel, etc.) to a cell expressing MT-SP1. In one preferred
embodiment, chimeric molecules are used to deliver the effector to
the cancer cell (or proliferating endothelial cell participating in
angiogeneisis).
[0249] In a chimeric molecule, two or more molecules that exist
separately in their native state are joined together to form a
single molecule having the desired functionality of all of its
constituent molecules. Typically, one of the constituent molecules
of a chimeric molecule is a "targeting molecule". The targeting
molecule is a molecule such as a ligand or an antibody that
specifically binds to its corresponding target, in this case an
MT-SP1 protein.
[0250] Another constituent of the chimeric molecule is an
"effector". The effector molecule refers to a molecule or group of
molecules that is to be specifically transported to the target cell
(e.g., a cell expressing an MT-SP1 polypeptide). The effector
molecule typically has a characteristic activity that is desired to
be delivered to the target cell. Effector molecules include, but
are not limited to cytotoxins, labels, radionuclides, ligands,
antibodies, drugs, liposomes, and the like.
[0251] In particular, where the effector component is a cytotoxin,
the chimeric molecule may act as a potent cell-killing agent
specifically targeting the cytotoxin to cells bearing a particular
target molecule. For example, chimeric fusion proteins which
include interleukin 4 (IL-4) or transforming growth factor
(TGF.alpha.) fused to Pseudomonas exotoxin (PE) or interleukin 2
(IL-2) fused to Diphtheria toxin (DT) have been shown to
specifically target and kill cancer cells (Pastan et al., Ann. Rev.
Biochem., 61: 331-354 (1992)).
[0252] A) The Targeting Molecule.
[0253] In a preferred embodiment, in the methods and compositions
of this invention, the targeting molecule is an antibody that
specifically binds to a MT-SP1 protein or to a fragment thereof.
The antibody can be a full-length antibody polyclonal or monoclonal
antibody, an antibody fragment (e.g. Fv, Fab, etc.), or a single
chain antibody (e.g. scFv).
[0254] The antibody can be produced according to standar methods
well known to those of skill in the art as described above. The
antibody once produced can be chemically conjugated to the
effector.
[0255] Where one of the effector molecule(s) is a protein, the
antibody can be a single chain antibody and the chimeric molecule
can be a recombinantly expressed fusion protein. Means of producing
such recombinant fusion proteins are well known to those of skill
in the art.
[0256] B) The Effector Molecule.
[0257] As described above, the effector molecule component of the
chimeric molecules of this invention may be any molecule whose
activity it is desired to deliver to cells that express or
overexpress a MT-SP1 protein. Particularly preferred effector
molecules include cytotoxins such as Pseudomonas exotoxin, or
Diphtheria toxin, radionuclides, radio-sensitizing agents, ligands
such as growth factors, antibodies, detectable labels such as
fluorescent, radio-opaque, or radioactive labels, and therapeutic
compositions such as liposomes and various drugs.
[0258] 1) Cytotoxins.
[0259] Particularly preferred cytotoxins include Pseudomonas
exotoxins, Diphtheria toxins, ricin, and abrin. Pseudomonas
exotoxin and Dipthteria toxin are most preferred.
[0260] Pseudomonas exotoxin A (PE) is an extremely active monomeric
protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells
through the inactivation of elongation factor 2 (EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP
ribosyl moiety of oxidized NAD onto EF-2).
[0261] The toxin contains three structural domains that act in
concert to cause cytotoxicity. Domain Ia (amino acids 1-252)
mediates cell binding. Domain II (amino acids 253-364) is
responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2, which inactivates the protein and causes cell death. The
function of domain Ib (amino acids 365-399) remains undefined,
although a large part of it, amino acids 365-380, can be deleted
without loss of cytotoxicity. See Siegall et al., J. Biol. Chem.
264: 14256-14261 (1989).
[0262] Where the targeting molecule (e.g. anti-MT-SP1) is fused to
PE, a preferred PE molecule is one in which domain Ia (amino acids
1 through 252) is deleted and amino acids 365 to 380 have been
deleted from domain Ib. However all of domain Ib and a portion of
domain II (amino acids 350 to 394) can be deleted, particularly if
the deleted sequences are replaced with a linking peptide such as
GGGGS.
[0263] In addition, the PE molecules can be further modified using
site-directed mutagenesis or other techniques known in the art, to
alter the molecule for a particular desired application. Means to
alter the PE molecule in a manner that does not substantially
affect the functional advantages provided by the PE molecules
described here can also be used and such resulting molecules are
intended to be covered herein.
[0264] For maximum cytotoxic properties of a preferred PE molecule,
several modifications to the molecule are recommended. An
appropriate carboxyl terminal sequence to the recombinant molecule
is preferred to translocate the molecule into the cytosol of target
cells. Amino acid sequences which have been found to be effective
include, REDLK (as in native PE), REDL, RDEL, or KDEL, repeats of
those, or other sequences that function to maintain or recycle
proteins into the endoplasmic reticulum, referred to here as
"endoplasmic retention sequences". See, for example, Chaudhary et
al. (1991) Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam et
al, J. Biol. Chem. 266: 17376-17381. Preferred forms of PE comprise
the PE molecule designated PE38QQR. (Debinski et al. Bioconj.
Chem., 5: 40 (1994)), and PE4E (see, e.g., Chaudhary et al. (1995)
J. Biol. Chem., 265: 16306). The targeting molecule (e.g.
anti-MT-SP1) may also be inserted at a point within domain III of
the PE molecule or into domain Ib. Methods of cloning genes
encoding PE fused to various ligands are well known to those of
skill in the art (see, e.g., Siegall et al., FASEB J., 3: 2647-2652
(1989); and Chaudhary et al. Proc. Natl. Acad. Sci. USA, 84:
45384542 (1987)).
[0265] Like PE, diphtheria toxin (DT) kills cells by
ADP-ribosylating elongation factor 2 thereby inhibiting protein
synthesis. Diphtheria toxin, however, is divided into two chains, A
and B, linked by a disulfide bridge. In contrast to PE, chain B of
DT, which is on the carboxyl end, is responsible for receptor
binding and chain A, which is present on the amino end, contains
the enzymatic activity (Uchida et al., Science, 175: 901-903
(1972); Uchida et al. J. Biol. Chem., 248: 3838-3844 (1973)).
[0266] In a preferred embodiment, the targeting molecule-Diphtheria
toxin fusion proteins of this invention have the native
receptor-binding domain removed by truncation of the Diphtheria
toxin B chain. Particularly preferred is DT388, a DT in which the
carboxyl terminal sequence beginning at residue 389 is removed.
Chaudhary, et al., Bioch. Biophys. Res. Comm., 180: 545-551 (1991).
Like the PE chimeric cytotoxins, the DT molecules may be chemically
conjugated to the MT-SP1 antibody, but, in a preferred embodiment,
the targeting molecule will be fused to the Diphtheria toxin by
recombinant means (see, e.g., Williams et al. (1990) J. Biol. Chem.
265: 11885-11889).
[0267] 2) Detectable Labels.
[0268] Detectable labels suitable for use as the effector molecule
component of the chimeric molecules of this invention include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include magnetic beads (e.g.
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
texas red, rhodamine, green fluorescent protein, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0269] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0270] 3) Ligands.
[0271] The effector molecule may also be a ligand or an antibody.
Particularly preferred ligand and antibodies are those that bind to
surface markers on immune cells. Chimeric molecules utilizing such
antibodies as effector molecules act as bifunctional linkers
establishing an association between the immune cells bearing
binding partner for the ligand or antibody and the tumor cells
expressing the MT-SP1 protein. Suitable antibodies and growth
factors are known to those of skill in the art and include, but are
not limited to, IL-2, IL-4, IL-6, IL-7, tumor necrosis factor
(TNF), anti-Tac, TGF.alpha., and the like.
[0272] 4) Other Therapeutic Moieties.
[0273] Other suitable effector molecules include pharmacological
agents or encapsulation systems containing various pharmacological
agents. Thus, the targeting molecule of the chimeric molecule may
be attached directly to a drug that is to be delivered directly to
the tumor. Such drugs are well known to those of skill in the art
and include, but are not limited to, doxirubicin, vinblastine,
genistein, an antisense molecule, and the like.
[0274] Alternatively, the effector molecule may be an encapsulation
system, such as a viral capsid, a liposome, or micelle that
contains a therapeutic composition such as a drug, a nucleic acid
(e.g. an antisense nucleic acid), or another therapeutic moiety
that is preferably shielded from direct exposure to the circulatory
system. Means of preparing liposomes attached to antibodies are
well known to those of skill in the art. See, for example, U.S.
Pat. No. 4,957,735, Connor et al., Pharm. Ther., 28: 341-365
(1985)
[0275] C) Attachment of the Targeting Molecule to the Effector
Molecule.
[0276] One of skill will appreciate that the MT-SP1 targeting
molecule and effector molecules may be joined together in any
order. Thus, where the targeting molecule is a polypeptide, the
effector molecule may be joined to either the amino or carboxy
termini of the targeting molecule. The targeting molecule may also
be joined to an internal region of the effector molecule, or
conversely, the effector molecule may be joined to an internal
location of the targeting molecule, as long as the attachment does
not interfere with the respective activities of the molecules.
[0277] The targeting molecule and the effector molecule may be
attached by any of a number of means well known to those of skill
in the art. Typically the effector molecule is conjugated, either
directly or through a linker (spacer), to the targeting molecule.
However, where both the effector molecule and the targeting
molecule are polypeptides it is preferable to recombinantly express
the chimeric molecule as a single-chain fusion protein.
[0278] 1) Conjugation of the Effector Molecule to the Targeting
Molecule.
[0279] In one embodiment, the targeting molecule (e.g.,
anti-MT-SP1Ab) is chemically conjugated to the effector molecule
(e.g., a cytotoxin, a label, a ligand, or a drug or liposome).
Means of chemically conjugating molecules are well known to those
of skill.
[0280] The procedure for attaching an agent to an antibody or other
polypeptide targeting molecule will vary according to the chemical
structure of the agent. Polypeptides typically contain variety of
functional groups; e.g., carboxylic acid (COOH) or free amine
(--NH.sub.2) groups, which are available for reaction with a
suitable functional group on an effector molecule to bind the
effector thereto.
[0281] Alternatively, the targeting molecule and/or effector
molecule may be derivatized to expose or attach additional reactive
functional groups. The derivatization may involve attachment of any
of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford Ill.
[0282] A "linker", as used herein, is a molecule that is used to
join the targeting molecule to the effector molecule. The linker is
capable of forming covalent bonds to both the targeting molecule
and to the effector molecule. Suitable linkers are well known to
those of skill in the art and include, but are not limited to,
straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Where the targeting molecule and the
effector molecule are polypeptides, the linkers may be joined to
the constituent amino acids through their side groups (e.g.,
through a disulfide linkage to cysteine). However, in a preferred
embodiment, the linkers will be joined to the alpha carbon amino
and carboxyl groups of the terminal amino acids.
[0283] A bifunctional linker having one functional group reactive
with a group on a particular agent, and another group reactive with
an antibody, may be used to form the desired immunoconjugate.
Alternatively, derivatization may involve chemical treatment of the
targeting molecule, e.g., glycol cleavage of the sugar moiety of a
the glycoprotein antibody with periodate to generate free aldehyde
groups. The free aldehyde groups on the antibody may be reacted
with free amine or hydrazine groups on an agent to bind the agent
thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation
of free sulfhydryl groups on polypeptide, such as antibodies or
antibody fragments, are also known (See U.S. Pat. No.
4,659,839).
[0284] Many procedure and linker molecules for attachment of
various compounds including radionuclide metal chelates, toxins and
drugs to proteins such as antibodies are known (see, e.g., European
Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and
4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47:
4071-4075). In particular, production of various immunotoxins is
well-known within the art and can be found, for example in
"Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,"
Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic
Press, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S.
Pat. Nos. 4,545,985 and 4,894,443.
[0285] In some circumstances, it is desirable to free the effector
molecule from the targeting molecule when the chimeric molecule has
reached its target site. Therefore, chimeric conjugates comprising
linkages which are cleavable in the vicinity of the target site may
be used when the effector is to be released at the target site.
Cleaving of the linkage to release the agent from the antibody may
be prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in
the vicinity of the target site. When the target site is a tumor, a
linker which is cleavable under conditions present at the tumor
site (e.g. when exposed to tumor-associated enzymes or acidic pH)
may be used.
[0286] A number of different cleavable linkers are known to those
of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and
4,625,014. The mechanisms for release of an agent from these linker
groups include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example,
includes a description of immunoconjugates comprising linkers which
are cleaved at the target site in vivo by the proteolytic enzymes
of the patient=s complement system. In view of the large number of
methods that have been reported for attaching a variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs,
toxins, and other agents to antibodies one skilled in the art will
be able to determine a suitable method for attaching a given agent
to an antibody or other polypeptide.
[0287] 2) Production of Fusion Proteins.
[0288] Where the MT-SP1 targeting molecule and/or the effector
molecule is relatively short (i.e., less than about 50 amino acids)
they may be synthesized using standard chemical peptide synthesis
techniques. Where both molecules are relatively short the chimeric
molecule may be synthesized as a single contiguous polypeptide.
Alternatively the targeting molecule and the effector molecule may
be synthesized separately and then fused by condensation of the
amino terminus of one molecule with the carboxyl terminus of the
other molecule thereby forming a peptide bond. Alternatively, the
targeting and effector molecules may each be condensed with one end
of a peptide spacer molecule thereby forming a contiguous fusion
protein.
[0289] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2:
Special Methods in Peptide Synthesis, Part A., Merrifield, et al.
J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid
Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
(1984).
[0290] In a preferred embodiment, the chimeric fusion proteins of
the present invention are synthesized using recombinant DNA
methodology. Generally this involves creating a DNA sequence that
encodes the fusion protein, placing the DNA in an expression
cassette under the control of a particular promoter, expressing the
protein in a host, isolating the expressed protein and, if
required, renaturing the protein.
[0291] DNA encoding the fusion proteins (e.g. anti-MT-SP1-PE38QQR)
of this invention may be prepared by any suitable method,
including, for example, cloning and restriction of appropriate
sequences or direct chemical synthesis by methods such as the
phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99
(1979); the phosphodiester method of Brown et al., Meth. Enzymol.
68: 109-151 (1979); the diethylphosphoramidite method of Beaucage
et al., Tetra. Lett., 22: 1859-1862 (1981); and the solid support
method of U.S. Pat. No. 4,458,066.
[0292] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0293] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0294] In a preferred embodiment, DNA encoding fusion proteins of
the present invention may be cloned using DNA amplification methods
such as polymerase chain reaction (PCR). Thus, for example, the
nucleic acid encoding an anti-MT-SP1 is PCR amplified, using a
sense primer containing the restriction site for NdeI and an
antisense primer containing the restriction site for HindIII. This
produces a nucleic acid encoding the anti-MT-SP1 sequence and
having terminal restriction sites. A PE38QQR fragment may be cut
out of the plasmid pWDMH4-38QQR or plasmid pSGC242FdN1 described by
Debinski et al. (1994) Int. J. Cancer, 58: 744-748. Ligation of the
anti-MT-SP1 and PE38QQR sequences and insertion into a vector
produces a vector encoding anti-MT-SP1 joined to the amino terminus
of PE38QQR (position 253 of PE). The two molecules are joined by a
three amino acid junction consisting of glutamic acid, alanine, and
phenylalanine introduced by the restriction site.
[0295] While the two molecules are preferably essentially directly
joined together, one of skill will appreciate that the molecules
may be separated by a peptide spacer consisting of one or more
amino acids. Generally the spacer will have no specific biological
activity other than to join the proteins or to preserve some
minimum distance or other spatial relationship between them.
However, the constituent amino acids of the spacer may be selected
to influence some property of the molecule such as the folding, net
charge, or hydrophobicity.
[0296] The nucleic acid sequences encoding the fusion proteins may
be expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and various higher eukaryotic cells such as
the COS, CHO and HeLa cells lines and myeloma cell lines. The
recombinant protein gene will be operably linked to appropriate
expression control sequences for each host. For E. coli this
includes a promoter such as the T7, trp, or lambda promoters, a
ribosome binding site and preferably a transcription termination
signal. For eukaryotic cells, the control sequences will include a
promoter and preferably an enhancer derived from immunoglobulin
genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence,
and may include splice donor and acceptor sequences.
[0297] The plasmids of the invention can be transferred into the
chosen host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0298] Once expressed, the recombinant fusion proteins can be
purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like (see, generally,
R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982),
Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification, Academic Press, Inc. N.Y. (1990)). Substantially pure
compositions of at least about 90 to 95% homogeneity are preferred,
and 98 to 99% or more homogeneity are most preferred for
pharmaceutical uses. Once purified, partially or to homogeneity as
desired, the polypeptides may then be used therapeutically.
[0299] One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the MT-SP1
targeted fusion protein may possess a conformation substantially
different than the native conformations of the constituent
polypeptides. In this case, it may be necessary to denature and
reduce the polypeptide and then to cause the polypeptide to re-fold
into the preferred conformation. Methods of reducing and denaturing
proteins and inducing re-folding are well known to those of skill
in the art (See, Debinski et al. (1993) J. Biol. Chem., 268:
14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:
581-585; and Buchner, et al. (1992) Anal. Biochem., 205:
263-270).
[0300] One of skill would recognize that modifications can be made
to the MT-SP1 targeted fusion proteins without diminishing their
biological activity. Some modifications may be made to facilitate
the cloning, expression, or incorporation of the targeting molecule
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
[0301] The compositions containing the present fusion proteins or a
cocktail thereof (i.e., with other proteins) can be administered
for therapeutic treatments. In therapeutic applications,
compositions are administered to a patient suffering from a
disease, e.g., a cancer, in an amount sufficient to cure or at
least partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health.
[0302] Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
[0303] It will be appreciated by one of skill in the art that there
are some regions that are not heavily vascularized or that are
protected by cells joined by tight junctions and/or active
transport mechanisms which reduce or prevent the entry of
macromolecules present in the blood stream. Thus, for example,
systemic administration of therapeutics to treat gliomas, or other
brain cancers, is constrained by the blood-brain barrier which
resists the entry of macromolecules into the subarachnoid
space.
[0304] One of skill in the art will appreciate that in these
instances, the therapeutic compositions of this invention can be
administered directly to the tumor site. Thus, for example, brain
tumors (e.g., gliomas) can be treated by administering the
therapeutic composition directly to the tumor site (e.g., through a
surgically implanted catheter). Where the fluid delivery through
the catheter is pressurized, small molecules (e.g. the therapeutic
molecules of this invention) will typically infiltrate as much as
two to three centimeters beyond the tumor margin.
[0305] Alternatively, the therapeutic composition can be placed at
the target site in a slow release formulation. Such formulations
can include, for example, a biocompatible sponge or other inert or
resorbable matrix material impregnated with the therapeutic
composition, slow dissolving time release capsules or
microcapsules, and the like.
[0306] Typically the catheter or time release formulation will be
placed at the tumor site as part of a surgical procedure. Thus, for
example, where major tumor mass is surgically removed, the
perfusing catheter or time release formulation can be emplaced at
the tumor site as an adjunct therapy. Of course, surgical removal
of the tumor mass may be undesired, not required, or impossible, in
which case, the delivery of the therapeutic compositions of this
invention may comprise the primary therapeutic modality.
[0307] E) Tumor Imaging and Radio-Sensitizing Compositions.
[0308] 1) Imaging Compositions.
[0309] In certain embodiments, the chimeric molecules of this
invention can be used to direct detectable labels to a tumor site.
This can facilitate tumor detection and/or localization. In a
particularly preferred embodiment, the effector component of the
chimeric molecule is a "radiopaque" label, e.g. a label that can be
easily visualized using x-rays. Radiopaque materials are well known
to those of skill in the art. The most common radiopaque materials
include iodide, bromide or barium salts. Other radiopaque materials
are also known and include, but are not limited to organic bismuth
derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque
polyurethanes (see U.S. Pat. No. 5,346,9810, organobismuth
composites (see, e.g., U.S. Pat. No. 5,256,334), radiopaque barium
polymer complexes (see, e.g., U.S. Pat. No. 4,866,132), and the
like.
[0310] The anti-MT-SP1 antibodie(s) can be coupled directly to the
radiopaque moiety or they can be attached to a "package" (e.g. a
liposome, a polymer microbead, etc.) carrying or containing the
radiopaque material.
[0311] 2) Radiosensitizers.
[0312] In another embodiment, the effector can be a radiosensitizer
that enhances the cytotoxic effect of ionizing radiation (e.g.,
such as might be produced by .sup.60Co or an x-ray source) on a
cell. Numerous radiosensitizing agents are known and include, but
are not limited to benzoporphyrin derivative compounds (see, e.g.,
U.S. Pat. No. 5,945,439), 1,2,4-benzotriazine oxides (see, e.g.,
U.S. Pat. No. 5,849,738), compounds containing certain diamines
(see, e.g., U.S. Pat. No. 5,700,825), BCNT (see, e.g., U.S. Pat.
No. 5,872,107), radiosensitizing nitrobenzoic acid amide
derivatives (see, e.g., U.S. Pat. No. 4,474,814), various
heterocyclic derivatives (see, e.g., U.S. Pat. No. 5,064,849),
platinum complexes (see, e.g., U.S. Pat. No. 4,921,963), and the
like.
[0313] The anti-MT-SP1 antibodie(s) can be coupled directly to the
radiopaque moiety or they can be attached to a "package" (e.g. a
liposome, a polymer microbead, etc.) carrying or containing the
radiosensitizing material.
VIII. Kits.
[0314] In still another embodiment, this invention provides kits
for practice of the assays or use of the therapeutics and/or
diagnostics described herein. In one preferred embodiment, the kits
comprise one or more containers containing antibodies and/or
nucleic acid probes and/or substrates suitable for detection of
MT-SP1 proteins or protein fragments, and/or MT-SP1 nucleic
acid(s), and/or and MT-SP1 protein activity, respectively. In other
embodiments, the kits include one or more of the MT-SP1 directed
chimeric molecules discussed herein. The kits may optionally
include any reagents and/or apparatus to facilitate practice of the
assays or delivery of the molecules described herein. Such reagents
include, but are not limited to buffers, pharmacological
excipients, labels, labeled antibodies, labeled nucleic acids,
filter sets for visualization of fluorescent labels, blotting
membranes, and the like.
[0315] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
assay methods or use of the chimeric molecules of this invention.
Preferred instructional materials provide protocols for assaying
MT-SP1 gene expression, and/or protein levels, and/or MT-SP1
protein activity, while other preferred instructional materials
provide guidance and instructions for the use of the chimeric
molecules described herein. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
EXAMPLES
[0316] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Reverse Biochemistry: Using Macromolecular Protease Inhibitors to
Identify a Membrane-Type Serine Protease in Epithelial Cancer and
Normal Tissue
[0317] This example describes the use of a "fold-specific"
inhibitor (.sup.i, .sup.ii) in studying the role of these
chymotrypsin-fold serine proteases in cancer. Ecotin or engineered
versions of ecotin are introduced into complex biological systems
as probes of proteolysis by these chymotrypsin-fold proteases.
When, as demonstrated herein, effects are observed upon treatment
with these unique inhibitors, then the large body of knowledge
concerning the biochemistry of these proteases can be tapped to
understand the structure and function of the target proteases.
[0318] For example, the molecular cloning, structural modeling, and
mechanistic understanding of the enzymes are immediately
accessible. Analogous to "reverse genetics" we refer to this
approach as "reverse biochemistry" and have applied it to identify
specific serine proteases in prostate cancer.
[0319] One useful model system for studying many issues that are
pertinent to prostate cancer is the development of the rodent
ventral prostate (VP) in explant cultures. Macromolecular
inhibitors of serine proteases of the chymotrypsin fold, ecotin and
ecotin M84R/M85R (see copending application Ser. Nos. 09/290,513
and 09/289,830, both filed on Apr. 12, 1999), inhibit ductal
branching morphogenesis and differentiation of the explanted rat
VP. Ecotin M84R/M85R is an 2800-fold more potent inhibitor of uPA
compared to ecotin (1 mM and 2.8 .mu.M respectively). However,
inhibition of prostate differentiation was seen with both
inhibitors, suggesting that uPA and other related serine proteases
are involved in the differentiation and continued growth of the rat
VP. Thus unidentified serine proteases may play a role in growth
and prevention of apoptosis in prostate epithelial cells in this
system.
[0320] Another well characterized model that is derived from human
prostate cancer epithelial cells is the PC-3 cell line (Kaighn et
al. (1979) Invest. Urology 17: 16-23). The PC-3 cell line expresses
uPA as assayed by enzyme-linked immunosorbent assay (ELISA) and by
Northern blotting of PC-3 mRNA (Yoshida et al. (1994) Cancer Res.
54: 3300-3304). We found that the primary tumor size in PC-3
implanted nude mice was significantly smaller in ecotin M84R/M85R
and ecotin wild-type treated mice treated for seven weeks compared
to the primary tumor size of PBS-treated mice after four weeks.
Metastasis from the primary tumors similarly were similarly lower
in the inhibitor-treated mice compared to PBS treated mice.
Inhibition was not unexpected with ecotin M84R/M85R treatment,
since uPA has been implicated in metastasis. However, wild-type
ecotin is a poor, micromolar inhibitor of uPA; one interpretation
of the data is that the decrease in tumor size and metastasis in
the mouse model involves the inhibition of additional serine
proteases. Thus identification of the serine proteases expressed by
PC-3 prostate cells may provide insight into the role of these
proteases in cancer and prostate growth and development. In this
example we have extended the strategy of using the polymerase chain
reaction (PCR) with degenerate oligonucleotide primers that were
designed using conserved sequence homology (Sakanari et al. (1989)
Proc. Natl. Acad. Sci. USA 86: 48634867; Wiegand et al. (1993) Gene
136: 167-175, Kang et al. (1992) Gene 110: 181-187) to identify
additional serine proteases made by cancer cells. Five independent
serine protease cDNAs derived from PC-3 mRNA were sequenced,
including a novel serine protease, which we refer to as
membrane-type serine protease 1 (MT-SP1), and the cloning and
characterization of this cDNA that encodes a mosaic, transmembrane
protease is reported.
Materials and Methods
[0321] Materials
[0322] All primers used were synthesized on a Applied Biosystems
391 DNA synthesizer. All restriction enzymes were purchased from
New England Biolabs. Automated DNA sequencing was carried out on an
Applied Biosystems 377 Prism sequencer, and manual DNA sequencing
was carried out under standard conditions. N-terminal amino acid
sequencing was performed on an ABI 477A by the Biomolecular
resource center. The synthetic substrates, Suc-AAPX-pNA,
[N-succinyl-alanyl-alanyl-prolyl-Xxx-pNA (Xxx=alanyl, aspartyl,
glutamyl, phenylalanyl, leucinyl, methionyl, and arginyl)], and
H-Arg-pNA, (arginyl-pNA), were purchased from Bachem.
Deglycosylation was performed using PNGase F (NEB). All other
reagents were of the highest quality available and purchased from
Sigma or Fisher unless otherwise noted.
[0323] Isolation of cDNA from PC-3 Cells
[0324] mRNA was isolated from PC-3 cells using the polyATtract
System 1000 kit (Promega). Reverse transcription was primed using
the "lock-docking" oligo dT primer (Borsont et al. (1992) PCR Meth.
Appl. 2: 144-148). Superscript II reverse transcriptase (Life
Technologies) was used in accordance with the manufacturer's
instructions to synthesize the cDNA from the PC-3 mRNA.
[0325] Amplification of MT-SP1 Gene
[0326] The degenerate primers used for amplifying the protease
domains were designed from the consensus sequences flanking the
catalytic histidine (5' His-primer) and the catalytic serine (3'
Ser-primer), similar to those described (Sakanari et al. (1989)
Proc. Natl. Acad. Sci. USA 86, 4863-4867). The 5' primer used is as
follows: 5'-TGG (AG)TI (CAG)TI (AT)(GC)I GCI (GA)CI CA(CT) TG-3'
(SEQ ID NO: 3), where nucleotides in parentheses represent
equimolar mixtures, and I represents deoxyinosine. This primer
encodes at least the following amino acid sequence: W (IV)
(I/V/L/M) (S/T) A (A/T) H C (SEQ ID NO: 4). The 3' primer used is
as follows: 5'-IGG ICC ICC I(GC)(AT) (AG)TC ICC (CT)TI (GA)CA
IG(ATC) (GA)TC-3' (SEQ ID NO: 5). The reverse complement of the 3'
primer encodes at least the following amino acid sequence: D
(A/S/T) C (K/E/Q/H) G D S G G P (SEQ ID NO: 6).
[0327] Direct amplification of serine protease cDNA was not
possible using the above primers. Instead, the first PCR was
performed with the 5'-His-primer and the oligo dT primer described
above, using the "touchdown" PCR protocol (Don et al. (1991)
Nucleic Acids Res. 19: 4008), with annealing temperatures
decreasing from 52.degree. C. to 42.degree. C. over 22 rounds, and
13 final rounds at 54.degree. C. annealing temperature. Cycle times
were 1 minute denaturing, 1 minute annealing, and 2 minute
extension times, followed by one final extension time of 15 minutes
after the final round of PCR. The template for the second PCR was
0.5 .mu.L (total reaction volume 50 .mu.L) of a 1 to 10 dilution of
the first PCR reaction mixture that was performed with the 5'
His-primer and the oligo dT. The second PCR reaction was primed
with the 5' His and the 3' Ser-primers and performed using the
touchdown protocol described above. All PCR reactions used 12.5
pmol of primer for 50 .mu.L reaction volume.
[0328] The product of the second reaction was purified on a 2%
agarose gel, and all products between 400 and 550 base pairs were
cut from the gel and extracted using the Qiaquick gel extraction
kit (Qiagen). These products were digested with the BamHI
restriction enzyme to cut any uPA cDNA, and all 400-500 bp
fragments were repurified on a 2% agarose gel. These reaction
products were subjected to a third PCR using the 5' His-primer and
the 3' Ser-primer using the identical touchdown procedure. These
reaction products were gel purified and directly cloned into the
pPCR2.1 vector using the TOPO TA ligation kit (Invitrogen). DNA
sequencing of the inserts determined the cDNA sequence from
nucleotides 1984-2460, see FIG. 1.
[0329] Northern Blot Analysis
[0330] .sup.32P-Labeled nucleotides were purchased from Amersham
Life Sciences. A cDNA fragment containing nucleotides 1173-2510 was
digested from EST w39209 using restriction enzymes EcoRI and BsmbI,
yielding a 1.3 kb nucleotide insert. Labeled cDNA probes were
synthesized using the Rediprime random primer labelling kit
(Amersham) and 20 ng of the purified insert. Poly(A)+RNA membranes
for Northern blotting were purchased from Origene (HB-1002,
HB-1018) and Clontech (Human II #7759-1, Human Cancer Cell Line
#7757). The blots were performed under stringent annealing
conditions as described in (Ausubel et al. (ed.). (1990) Current
protocols in molecular biology. Wiley & Sons, New York,
N.Y.).
[0331] Construction of Expression Vectors
[0332] The mature protease domain and a small portion of the pro
domain (nucleotides 1822-2601) cDNA were amplified using PCR from
EST w39209 and ligated into the pQE30 vector (Qiagen). This
construct is designed to overexpress the protease sequence from
amino acids (aa) 596-855 with the following fusion:
Met-Arg-Gly-Ser-His.sub.6-aa596-855. The His-tag fission allows
affinity purification using metal chelate chromatography. The
change from Ser.sup.805, encoded by TCC, to Ala (GCT) was performed
using PCR. The presence of the correct Ser to Ala substitution in
the pQE30 vector was verified by DNA sequence analysis.
[0333] Expression and Purification of the Protease Domain
[0334] The above-mentioned plasmids were separately transformed
into E. coli X-90 to afford high-level expression of recombinant
protease gene products (Evnin et al. (1990) Proc. Natl. Acad. Sci.
USA 87, 6659-6663). Expression and purification of the recombinant
enzyme from solubilized inclusion bodies was performed as described
previously (Unal et al. (1997) J. Virol. 71, 7030-7038). Protein
containing fractions were pooled and dialyzed overnight at
4.degree. C. against 50 mM Tris pH 8, 10% glycerol, 1
mM-mercaptoethanol, 3M urea. Autoactivation of the protease was
monitored upon dialysis against storage buffer (50 mM Tris pH 8,
10% glycerol) at 4.degree. C. using the substrate Spectrozyme tPA
(hexahydrotyrosyl-Gly-Arg-pNA, American Diagnostica). Hydrolysis of
Spectrozyme tPA was monitored at 405 nM for the formation of
p-nitroaniline using a UVIKON 860 spectrophotometer. Activated
protease was bound to an immobilized p-aminobenzamidine resin
(Pierce) that had been equilibrated with storage buffer. Bound
protease was eluted with 100 mM benzamidine and the protein
containing fractions were pooled. Excess benzamidine was removed
using FPLC with a Superdex 70 (Pharmacia) gel filtration column
that was equilibrated with storage buffer. Protein containing
fractions were pooled and stored at -80.degree. C. The cleavage of
the purified Serg.sup.805 Ala protease domain was performed at
37.degree. C. by addition of active recombinant protease domain to
10 nM. Cleavage was monitored by SDS-PAGE.
[0335] Determination of Substrate Kinetics
[0336] The purified serine protease domain was titrated with
4-methylumbelliferyl p-guanidinobenzoate (MUGB) to obtain an
accurate concentration of enzyme active sites (.sup.iii). Enzyme
activity was monitored at 25.degree. C. in assay buffer containing
50 mM Tris pH 8.8, 50 mM NaCl, and 0.01% Tween 20. The final
concentration of substrate Spectrozyme tPA ranged from 1 .mu.M400
.mu.M. Enzyme concentrations ranged from 40 pM-800 pM. Active site
titrations were performed on a Fluoromax-2 spectrofluorimeter.
Measurements were plotted using the KaleidaGraph program (Synergy,
Reading, Pa.), and the K.sub.m, k.sub.cat, and k.sub.cat/K.sub.m
for Spectrozyme tPA was determined using the Michaelis-Menten
equation.
[0337] Inhibition of MT-SP1 Protease Domain with Ecotin and Ecotin
M84R/M85R
[0338] Ecotin and ecotin M84R/M85R were purified from E. coli as
described in copending application Ser. Nos. 09/290,513 and
09/289,830, both filed on Apr. 12, 1999. Various concentrations of
ecotin or ecotin M84R/M85R were incubated with the His-tagged
serine protease domain in a total volume of 990 .mu.L of buffer
containing 50 mM NaCl, 50 mM Tris-HCl (pH 8.8), 0.01% Tween 20. 10
.mu.L of Spectrozyme tPA was added, yielding a solution containing
100 .mu.M substrate. The final enzyme concentration was 63 .mu.M,
and the ecotin and ecotin M84R/M85R concentration ranged from 0.1
nM to 50 nM. The data were fit to the equation derived for kinetics
of reversible tight-binding inhibitors (Morrison (1969) Biochim.
Biophys. Acta 185: 269-286, Williams and Morrison (1979) Methods.
Enzymol. 63: 437-467), and the values for apparent K.sub.i were
determined.
Results
[0339] Cloning of Serine Protease Domain cDNAs from PC-3 Cells and
Amplification of MT-SP1 cDNA
[0340] PCR amplification of serine protease cDNA was performed
using "consensus cloning", where the amplification was performed
with degenerate primers designed to anneal to cDNA encoding the
region about the conserved catalytic histidine (5' His-primer) and
the conserved catalytic serine (3' Ser-primer). The consensus
primers were designed using 37 human sequences within a sequence
alignment of 242 serine proteases of the chymotrypsin fold that are
reported in the Swiss protein database. In order to bias the screen
for previously unidentified proteases in the PC-3 cDNA, uPA cDNA
was cut and removed using the known BamHI endonuclease site in the
uPA cDNA sequence. The expected size of the cDNA fragments
amplified between His.sup.57 and Ser.sup.195 cDNA (standard
chymotrypsinogen numbering) is between 400-550 base pairs;
statistically, only one in ten cDNAs of that length will be cleaved
by BamHI. Thus, cDNAs obtained from the PCR reactions with the 5'
His-primer and 3' Ser-primer were size selected for the 400-550 bp
range, digested with BamHI and purified from any digested cDNAs.
After a subsequent round of PCR, the products were cloned into
pPCR2.1 (FIG. 2). Twenty clones were digested with EcoRI to monitor
the size of the cDNA insert. Three clones lacked inserts of the
correct size. The remaining seventeen clones containing inserts
between 400 and 550 bp were sequenced. Blast searches of the
resulting sequences revealed that six clones did not match serine
protease sequences. The remaining cDNAs yielded clones
corresponding to factor XII (2 clones), protein C (2 clones),
trypsinogen type IV (2 clones), uPA (1 clone) and a cDNA denoted as
membrane-type serine protease 1 (MT-SP1) (4 clones). Additional
serine protease sequences may not have been found because they were
digested by BamHI, lost in the size selection, or were present in
lower frequencies.
[0341] Multiple EST sequences were found for the cDNA. EST
accessions aa459076, aa219372, and w39209 were used extensively for
sequencing the cDNA starting from nucleotide 746, and 2461-3142,
but no start codon was observed. A sequence was also found in
GenBank, accession no. U20428. This sequence also lacks the 5' end
of the cDNA, but allowed amplification of cDNA from nucleotides
196-745. Rapid amplification of cDNA ends (RACE) techniques
(Frohman (1993) Methods Enzymol. 218: 340-356) were used to obtain
further 5' cDNA sequence. Application of RACE did not yield a clone
containing the entire 5' untranslated region, but the sequence
obtained contained a stop codon in frame with the Kozak start
sequence (Kozak (1991) J. Cell Biol. 115: 887-903), giving
confidence that the full coding-sequence of the cDNA has been
obtained. The nucleotide sequence and predicted amino acid sequence
are shown in FIG. 1 (SEQ ID NO: 1 and 2).
[0342] The nucleotide sequence surrounding the proposed start codon
matches the optimal sequence of ACCATGG (SEQ ID NO: 7) for
translation initiation sites proposed by Kozak (supra.). In
addition, there is a stop codon in frame with the putative start
codon, which gives further evidence that initiation occurs at that
site. The DNA sequence predicts an 855 amino acid mosaic protein
composed of multiple domains (FIG. 3). The coding sequence does not
contain a typical signal peptide, but does contain a single
hydrophobic sequence of 26 residues (residues 55-81), which is
flanked by a charged residue on each side. This sequence may
constitute a signal anchor (SA) sequence, similar to that observed
in other proteases, including hepsin (Leytus et al. (1988)
Biochemistry 27: 1067-1074) and enteropeptidase (Kitamoto et al.
(1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592). Following the
putative SA sequence are two CUB domains (Bork and Beckmann (1993)
J. Mol. Biol. 231: 539-545), which are named after the proteins in
which the modules were first discovered: complement subcomponents
C1s and C1r, urchin embryonic growth factor (Uegf), and bone
morphogenetic protein 1 (BMP1). CUB domains have conserved
characteristics, which include the presence of four cysteine
residues and various conserved hydrophobic and aromatic positions
(Bork and Beckmann (1993) J. Mol. Biol. 231: 539-545). The CUB
domain, which has recently been characterized crystallographically
(Varela et al. (1997) J. Mol. Biol. 274: 635-649), consists of
ten-strands that are organized into two 5-stranded .beta.-sheets.
Following the CUB domains are four LDLR repeats (Krieger and Herz
(1994) Annu. Rev. Biochem. 63: 601-637), which are named after the
receptor ligand-binding repeats that are present in the LDL
receptor. These repeats have a highly conserved pattern and spacing
of six cysteine residues that form three intramolecular disulfide
bonds. The final domain observed is the serine protease domain. The
alignments of these domains with other members of their respective
classes are shown in FIG. 4.
[0343] Tissue Distribution of MT-SP1 mRNA.
[0344] Northern blots of human poly(A)+ RNA, using a 1.3 kB
fragment of MT-SP1 cDNA fragment as a probe, show a .about.3.3 kB
fragment appearing in epithelial tissues including the prostate,
kidney, spleen, liver, leukocytes, lung, small intestine, stomach,
thymus, colon, and placenta, and explants of human breast cancer
and mastases. This band was not observed in muscle, brain, ovary,
or testis (FIG. 5). Similar experiments performed on a human cancer
cell line blot shows that MT-SP1 is expressed in the Colorectal
adenocarcinoma, SW480, and human breast cancer, but was not
observed in the Promyelocytic Leukemia HL-60, HeLa Cell S3, Chronic
Myelogenous Leukemia K-562, Lymphoblastic Leukemia MOLT-4,
Burkitt's Lymphoma Raji, Lung Carcinoma A549, or Melanoma G361
lanes (data not shown). MT-SP1 is also expressed in blood vessels
of prostatic and gastric cancers. This 3.3 kB mRNA fragment is
slightly longer than the 3.1 kB sequence presented in FIG. 5,
suggesting that there may still be further sequence in the 5'
untranslated region that has not been identified.
[0345] Activation and Purification of His-MT-SP1 Protease
Domain
[0346] The serine protease domain of MT-SP1 was expressed in E.
coli as a His-tagged fusion, and was purified from inclusion bodies
under denaturing conditions using metal-chelate affinity
chromatography. The yield of enzyme after this step was
approximately 3 mg of protein per liter of E. coli culture. This
denatured protein refolded when the urea was slowly dialyzed away
from the protein. Surprisingly, the purified renatured protein
showed a time dependent shift on an SDS-PAGE gel (FIG. 6A, lanes
(a) 1-7), with the lower fragment being the size of the mature,
processed enzyme, lacking the His tag. N-terminal sequencing of the
purified, activated protease domain yielded the expected VVGGT
activation sequence. When the refolded protein was tested for
activity using the synthetic substrate
hexahydrotyrosyl-glycyl-arginyl-paranitroanilide (Spectrozyme tPA),
a time dependent increase in activity was observed (FIG. 6B). In
contrast, the protease domain that contains the Ser.sup.805 Ala
mutation did not either show a change in size on an SDS
polyacrylamide gel or an increase in enzymatic activity under
identical conditions (data not shown), suggesting that the
catalytic serine is necessary for activation and not the result of
a contaminating protease. In order to show that the cleavage of the
protease domain was a result of His-tagged MT-SP1 protease
activity, the inactive Ser.sup.805 Ala protease domain was treated
with purified recombinant enzyme (FIG. 6C). This treatment results
in the formation of a cleavage product that corresponds to the size
of the active protease (FIG. 6C, lane 7). Untreated protease domain
does not get cleaved (FIG. 6C, lane 8). From these results, it is
concluded that the protease autoactivates upon refolding. The
activated protease was separated from inactive protein and other
contaminants using affinity chromatography with p-aminobenzamidine
resin. Purified protein was analyzed by SDS-PAGE and no other
contaminants were observed. Similarly, immunoblotting with
polyclonal antiserum against purified protease domain (raised in
rabbits at Berkeley Antibody Company) revealed one band. Under
non-reducing conditions, the pro-region is disulfide linked to the
protease domain; thus, this purified protein was also
immunoreactive with the monoclonal antibody (Qiagen) directed
against the amino-terminal Arg-Gly-Ser-His.sub.4 epitope that is
contained in the recombinant protease domain, further indicating
the purity and identity of the protein (data not shown).
[0347] Kinetic Properties of Purified His-MT-SP1 Protease
Domain
[0348] The enzyme concentration was determined using an active site
titration with MUGB. The catalytic activity of the protease domain
was monitored using pNA substrates. Purified protease domain was
tested for hydrolytic activity against tetrapeptide substrates of
the form Suc-AAPX-pNA, which contained various amino acids at the
P1 position (P1-Ala, Asp, Glu, Phe, Leu, Met, Lys, or Arg). The
only substrates with detectable activity were those with P1-Lys or
P1-Arg. The serine protease domain with the Ser.sup.805 Ala
mutation had no detectable activity. The activity of the protease
domain was further characterized using the substrate Spectrozyme
tPA (hexahydrotyrosyl-Gly-Arg-pNA), yielding: K.sub.m=31.4.+-.4.2
.mu.M, k.sub.cat=2.6.times.10.sup.2.+-.6.5 s.sup.-1, and
k.sub.cat/K.sub.m=6.9.times.10.sup.6.+-.2.3.times.10.sup.6
M.sup.-1s.sup.-1. Ecotin inhibition of the MT-SP1 His-tagged
protease domain fits a tight-binding reversible inhibitory model as
observed for ecotin interaction with other serine protease targets.
Inhibition assays using ecotin and ecotin M84R/M85R yielded
apparent K.sub.i's of 782.+-.92 pM and 9.8.+-.1.5 pM
respectively.
Discussion
[0349] Structural Motifs of MT-SP1
[0350] In this work, we characterize the expression of
chymotrypsin-fold proteases by PC-3 cells and cloned a member of
this family we call MT-SP1. The name membrane-type serine protease
1 (MT-SP1) is given to be consistent with the nomenclature of the
membrane-type matrix metalloproteases (MT-MMPs) (Nagase (1997)
Biol. Chem. 378, 151-160). The cDNA likely encodes a membrane-type
protein due to the lack of a signal sequence and the presence of a
putative signal anchor (SA) that is also seen in other
membrane-type serine proteases hepsin (Leytus et al. (1988)
Biochemistry 27: 1067-1074), enteropeptidase (Kitamoto et al.
(1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592), TMPRSS2
(Poloni-Giacobino et al. (1997) Genomics 44, 309-320), and human
airway trypsin-like protease (Yamakoka et al. (1998) J. Biol. Chem.
273, 11895-11901). We propose that proteins that are localized to
the membrane through a signal anchor and that encode a chymotrypsin
fold serine protease domain be categorized in the MT-SP family. The
membrane localization of MT-SP1 is supported by immunofluorescence
experiments that localize the protease domain to the extracellular
cell surface.
[0351] Following the putative SA are several domains that are
thought to be involved in protein-protein interactions or protein
ligand interactions. For example, CUB domains can mediate
protein-protein interactions, as with the seminal plasma
PSP-I/PSP-II heterodimer that is built by CUB domain interactions
and with procollagen C-proteinase enhancer protein and procollagen
C-proteinase (BMP-1) (Kessler and Adar (1989) Eur. J. Biochem. 186,
115-121; Hulmes et al. (1997) Matrix Biol. 16, 41-45).
Interestingly, most of the proteins that contain CUB domains are
involved in developmental processes or are involved in proteolytic
cascades, which suggests that MT-SP1 may play a similar role. The
four repeated motifs that follow the CUB domains are known as LDL
receptor ligand-binding repeats, named after the seven copies of
repeats found in the LDL receptor. There are several negatively
charged amino acids between the fourth and sixth cysteines that are
highly conserved in the LDL receptor and also seen in the LDLR
repeats of MT-SP1. The conserved motif Ser-Asp-Glu (residues 44-46
in FIG. 4) are known to be important for binding the positively
charged residues of the LDL receptor ligands apolipoprotein B-100
(ApoB-100) and ApoE. The ligand binding repeats of MT-SP1 most
likely do not mediate interaction with ApoB-100 or ApoE, but may be
involved in the interaction with other positively charged ligands.
For example, LDLR repeats in the LDL receptor-related protein have
been implicated the binding and recycling of protease/inhibitor
complexes such as uPA/plasminogen activator inhibitor-1 (PAI-1)
complexes (reviewed in Strickl et al. (1995) FASEB J. 9, 890-898;
Moestrup (1994) Biochim. Biopys. Acta 1197, 197-213). It also has
been shown that the pro domain of enteropeptidase is involved in
interactions with its substrate trypsinogen, allowing 520-fold
greater catalytic efficiency in the cleavage compared to the
protease domain alone (Lu et al. (1997) J. Biol. Chem. 272,
31293-31300). By analogy, similar interactions should occur between
MT-SP1 and its substrates. Thus, further investigation of MT-SP1
CUB domain or LDLR repeat interactions may yield insight into the
function of this protein.
[0352] The amino acid sequence of the serine protease domain of
MT-SP1 is highly homologous to other proteases found in the family
(FIG. 4). The essential features of a functional serine protease
are contained in the deduced amino acid sequence of the domain. The
residues that comprise the catalytic triad, His.sup.656,
Asp.sup.711, Ser.sup.805, corresponding to His.sup.57, Asp.sup.102,
and Ser.sup.195 in chymotrypsin, are observed in MT-SP1 (see Perona
and Craik (1995) Protein Sci. 4: 337-360, Perona and Craik (1997)
J. Biol. Chem. 272: 29987-29990 for reviews). The sequence
Ser.sup.214-Trp.sup.215-Gly.sup.216
(Ser.sup.825-Trp.sup.826-Gly.sup.827) which is thought to interact
with the side chains of the substrate for properly orienting the
scissile bond is present. Gly.sup.193 (Gly.sup.803) and Gly.sup.196
(Gly.sup.805), which are thought to be necessary for proper
orientation of Ser.sup.195 (Ser.sup.805) also are present. Based
upon homology to chymotrypsin, three disulfide bonds are predicted
to form within the protease domain at Cys.sup.44-Cys.sup.58,
Cys.sup.168-Cys.sup.182, and Cys.sup.191-Cys.sup.220
(Cys.sup.643-Cys.sup.657, Cys.sup.776-Cys.sup.790,
Cys.sup.801-Cys.sup.830), and a fourth disulfide bond should form
between the catalytic and the pro-domain Cys.sup.122-Cys.sup.1
(Cys.sup.731-Cys.sup.604), as observed for chymotrypsin. This
predicted disulfide with the pro-domain suggests that the active
catalytic domain should still be localized to the cell surface via
a disulfide linkage. The presence of the catalytic machinery and
other conserved structural components described above suggest that
all features necessary for proteolytic activity are present in the
encoded sequence.
[0353] Substrate Specificity of the MT-SP1 Protease Domain
[0354] The S1 site specificity (Schecter and Berger (1967) Biochem.
Biophys. Res. Commun. 27: 157-162) of a protease is largely
determined by the amino acid residue at position 189. This position
is occupied by an aspartate in MT-SP1, suggesting that the protease
has specificity for Arg/Lys in the P1 position. In addition, the
presence of a polar Gln.sup.192 (Gln.sup.803), as in trypsin is
consistent with basic specificity. Furthermore, the presence of
Gly.sup.216 (Gly.sup.827) and Gly.sup.226 (Gly.sup.837) is
consistent with the presence of a deep S1 pocket, unlike elastase,
which has Val.sup.216 and Thr.sup.226 that block the pocket and
thereby contribute to the P1 specificity for small hydrophobic side
chains. The specificity at the other subsites is largely dependent
upon the nature of the seven loops A-E and loops 2 and 3 (FIG. 4).
Loop C in enterokinase has a number of positively charged residues
that are thought to interact with the negatively charged activation
site in trypsinogen, Asp-Asp-Asp-Asp-Lys (SEQ ID NO:8). One known
substrate for MT-SP1 (as described below) is the activation site of
MT-SP1, which is Arg-Gln-Ala-Arg (residues 611-614). Loop C
contains two aspartate residues that may participate in the
recognition of the activation sequence.
[0355] One means of obtaining further data on substrate specificity
is by characterization of the activity of the recombinant
proteolytic domain. Enterokinase has been characterized from both
recombinant (LaVallie et al. (1993) J. Biol. Chem. 268:
23311-23317) and native (Light and Fonseca (1984) J. Biol. Chem.
259: 13195-13198; Matsushima et al. (1994) J. Biol. Chem. 269:
19976-19982) sources. However proteolytic activity for the other
reported membrane-type serine proteases hepsin (Leytus et al.
(1988) Biochemistry 27: 1067-1074) and TMPRSS2 (Poloni-Giacobino et
al. (1997) Genomics 44: 309-320) are only predicted based upon
sequence homology. In order to produce active recombinant MT-SP1, a
His-tagged fusion of the protease domain was cloned into an E. coli
vector and expressed and purified to homogeneity. Fortuitously, the
protease domain refolded and autoactivated after resuspension and
purification from inclusion bodies. This activity, coupled with the
lack of activity in the Ser.sup.195 Ala (Ser.sup.805 Ala) variant,
demonstrates that the cDNA encodes a catalytically proficient
protease. Autoactivation of the protease domain at the
arginine-valine site (Arg.sup.614-Val.sup.615) shows that the
protease has Arg/Lys specificity as predicted by the sequence
homology to other proteases of basic specificity. Specificity and
selectivity are confirmed by the lack of cleavage of AAPX-pNA
substrates that do not have X=R, K. Further characterization with
hexahydrotyrosyl-Gly-Arg-pNA (Spectrozyme tPA) revealed an active
enzyme with k.sub.cat=2.6.times.10.sup.2/s. However, the His-tagged
serine protease domain does not cleave H-Arg-pNA, showing that,
unlike trypsin, there is a requirement for additional subsite
occupation for catalytic activity. This suggests that the enzyme is
involved in a regulatory role that requires selective processing of
particular substrates rather than non-selective degradation.
[0356] MT-SP1 Function
[0357] In other studies, we have found that inhibition of serine
protease activity by ecotin or ecotin M84R/M85R inhibits
testosterone-induced branching ductal morphogenesis and enhances
apoptosis in a rat ventral prostate model. Moreover, the rat
homolog of MT-SP1 is expressed in the normal rat ventral prostate
(data not shown). Assays of the protease domain with ecotin and
ecotin M84R/M85R showed that the enzymatic activity is strongly
inhibited (782.+-.92 pM, 9.8.+-.1.5 pM respectively), suggesting
that rat MT-SP1 is likely to be inhibited at the concentrations of
these inhibitors used in our experiments. MT-SP1 inhibition may
result in the observed inhibition of differentiation and/or
increased apoptosis. Future studies are aimed at definitively
resolving the role of MT-SP1 in prostate differentiation. The broad
expression of MT-SP1 in epithelial tissues is consistent with the
possibility that it is involved in cell maintenance or growth and
differentiation, perhaps by activating growth factors or by
processing prohormones. Studies examining the direct role of MT-SP1
in differentiation and growth of the epithelium in glandular
tissues like the prostate are underway.
[0358] MT-SP1 may participate in a proteolytic cascade that results
in cell growth and or differentiation. Another structurally similar
membrane-type serine protease, enteropeptidase (FIG. 3), is
involved in a proteolytic cascade by which activation of
trypsinogen leads to activation of downstream intestinal proteases
(5). Enteropeptidase is expressed only in the enterocytes of the
proximal small intestine thus precisely restricting activation of
trypsinogen. Thus, in contrast to secreted proteases that may
diffuse throughout the organism, the membrane association of MT-SP1
should also allow the proteolytic activity to be precisely
localized, which may be important for proper physiological
function; improper localization of the enzyme or levels of
downstream substrates could lead to disease.
[0359] We have found subcutaneous coinjection of PC-3 cells with
wild-type ecotin or ecotin M84R/M85R led to a decrease in the
primary tumor size compared to animals in whom PC-3 cells and
saline were injected. Since wild-type ecotin is a poor, micromolar
inhibitor of uPA, serine proteases other than uPA likely are
involved in this primary tumor proliferation. Both wild-type ecotin
and ecotin M84R/M85R are potent, subnanomolar inhibitors of MT-SP1,
raising the possibility that MT-SP1 plays an important role in
progression of epithelial cancers expressing this protease.
[0360] Ecotin injected intraperitoneally also inhibited tumor
growth indicating that treatment by administration of MT-SP1
modulators can be accomplished using systemic administration.
[0361] Direct biochemical isolation of the substrates may be
possible if MT-SP1 adhesive domains such as the CUB domains or LDLR
repeats interact with the substrates. In addition, likely
substrates may be predicted and tested using knowledge of extended
enzyme specificity. For example, the characterization of the
substrate specificity of granzyme B allowed the prediction and
confirmation of substrates for this serine protease (Harris et al.
(1998) J. Biol. Chem. 273: 27364-27373). Thus, these complimentary
studies should further shed light on the physiological function of
this enzyme.
Example 2
[0362] Membrane-type serine protease 1 (MT-SP1) was identified as a
transmembrane protease expressed by a human prostate cancer cell
line, PC-3. We have examined the expression of MT-SP1 in gastric
cancer tissues and assessed the potential role of this protease in
cancer progression. Western blot and RT-PCR analysis demonstrated
exclusive expression of MT-SP1 in the cancer tissues of some cases.
Immunohistochemically, MT-SP1 was localized in cancer cells,
endothelial cells and leukocytes. Because the expression in
endothelial cells was especially intense, its labeling index (LI)
was calculated (0-98, 31.+-.5%). MT-SP1 LI was significantly higher
in specimens of poorly differentiated gastric cancer than in well
differentiated cancer specimens (46.+-.10, 15.+-.6%, respectively,
p<0.05). The 11 patients with high MT-SP1 expression >=40%)
had a lower survival rate than the 21 patients with low MT-SP1
expression (<40%) or the 9 patients without MT-SP1 expression
(p<0.05). These results suggest that MT-SP1 expression in
endothelial cells may play an important role in angiogenesis in
cancer tissues and is a significant prognostic factor in gastric
cancer.
[0363] Materials and Methods
[0364] Materials.
[0365] Surgical specimens of primary tumors obtained from 41
patients who underwent gastrectomy for gastric cancer between 1985
and 1995 at the Santa Clara Valley Medical Center, San Jose, Calif.
and the Palo Alto Veteranis Affair Medical Center, Palo Alto,
Calif., were subjected to immunohistochemical analysis. In
addition, surgically resected specimens of gastric cancer and
adjacent normal tissue from two patients at the National Defense
Medical College Hospital, Japan were used for Western and mRNA
analysis. Depth of tumor invasion, lymph node involvement, distant
metastases and pathologic characteristics were evaluated for each
tumor. TNM classification by the UICC was used for stage grouping
(Hermanek and Sobin (1992) UICC TNM classification of malignant
tumours, 4th ed. 2nd rev. edition. Berlin: Springer-Verlag). Tumor
histology was divided into three groups: well differentiated,
moderately differentiated and poorly differentiated
adenocarcinomas.
[0366] Western Immunoblotting Analysis.
[0367] We compared MT-SP1 expression in cancer tissue with that in
adjacent normal tissue, using immunoblotting analysis. Surgical
samples were homogenized in PBS (phosphate-buffered saline)
containing 0.1% Triton X-100. The extracts were electrophoresed on
10% SDS-polyacrylamide gels then electrically transferred to
polyvinylidene difluoride membrane. Membranes were treated with
nonfat milk to reduce nonspecific binding, then incubated for 1 h
at room temperature with the primary antibody. After extensive
washing, blots were incubated with peroxidase-conjugated second
antibodies and developed with detection reagents. Protein content
was measured using the BCA protein assay and bovine serum albumin
as the standard (Pierce, Rockford, Ill.).
[0368] RT-PCR.
[0369] mRNA was extracted from each specimen and purified using the
RNA STAT-60.TM. kit (Tel-test, Inc., Friendswood, Tex.). Tissues
were homogenized, lysed, and mRNA extracted and purified according
to the vendoris suggested protocol. mRNA was quantified by
measuring the spectroscopic absorbance at 260 and 280 nm. RT-PCR
was performed with the Titan.TM. one tube RT-PCR system according
to the manufacturer's protocol (Boehringer Mannheim, Indianapolis,
Ind.). One mg of template RNA was reverse transcribed to cDNA in 50
ml of reaction tube with 15 mM MgCl.sub.2, 0.2 mM deoxynucleotide
mix, 20 pmol of each MT-SP1 primer, enzyme mix at 50.degree. C. for
30 min. THE produced cDNA was directly amplified using a thermal
cycler. Initial denaturation was done at 94.degree. C. for 2 min
followed by 10 and 25 cycles of amplification. The first cycle
consisted of 30 s of denaturation at 94.degree. C., 30 s of
annealing at 55.degree. C., and 90 s for enzymatic primer extension
at 68.degree. C. the final extension was carried out at 68.degree.
C. for 7 min. The following oligonucleotides were used as RT-PCR
primers: MT-SP1-F: 5'-TGC GAC AGT GTG AAC GAC TGC GGA GAC AAC-3'
(SEQ ID NO: _); and MT-SP1-R: 5'-CTC CAC GCT GGA CAG GGG TCC CCC
GGA ATC-3' (SEQ ID NO: _).
[0370] As a positive control, we used RNA extract from PC-3 cells
(human prostate cancer cell line). Aliquots (10 mcl) of the RT-PCR
produce were electrophoresed in 1.5% agarose gel in 1.times.TAE (40
mM Tris acetate/2 mM sodium EDTA/glacial acetic acid, pH 8.8)
containing 0.5 .mu.g ethidium bromide.
[0371] Immunohistochemistry
[0372] Surgical specimens were preserved in a 10% neutralized
formaldehyde solution. Each block of paraffin-embedded tumor
specimen was cut into 5 mm sections and deparaffinized in xylene
and ethanol, then immersed in 3% hydrogen peroxide-methanol to
inhibit endogenous peroxidase. After treatment with normal goat
serum, the sections were incubated with a 1:100 dilution of rabbit
antihuman MT-SP1 antibody overnight at 4.degree. C. They were
washed then treated consecutively with a biotinylated goat
anti-rabbit antibody for 1 h. The 3,3'-diaminobenzidine substrate
kit and Vectastain Elite ABC kit (Vector Laboratories) were used
according to the manufacturer's suggested protocol. Counterstaining
was performed with hematoxylin. Negative controls for the
immunostaining were carried out by replacing the primary antibody
with preimmune rabbit immunoglobulin. Vessel rings were counted at
200.times. magnification. Ten fields were counted for each case,
and the proportion of total rings that were MT-SP1-positive was
defined as the MT-SP1 labeling index (MT-SP LI).
[0373] Statistics.
[0374] Results shown are the mean .+-.SE. Spearman rank correlation
analysis was used to correlate the degree of each histopathologic
factor with MT-SP LI. The cumulative survival rates were calculated
by the method of Kaplan-Meier. The survival rates for different
groups of patients were compared by the generalized Wilcoxon test.
The specific contribution of prognostic variables was examined by
means of a multivariate Coxis proportional hazard model. A p value
of less than 0.05 was considered statistically significant.
Results
[0375] Immunoblotting for MT-SP1.
[0376] Tissue from two cases of stomach cancer were analyzed by
Western immunoblotting using equal amounts (by weight) of cell
lysates from cancerous and adjacent normal stomach tissue. Using a
polyclonal antiserum produced against MT-SP1, the presence of two
bands (45 and 100 kD), most intense in the tumor tissue of case 2
(FIG. 1), were demonstrated. These two bands in tumor and adjacent
normal tissues from case 1 were less intense.
[0377] RT-PCR Analysis for MT-SP1.
[0378] Analysis of MT-SP1 mRNA from stomach tissues demonstrated
the expression in only tumor tissue of case 2 but not in that of
case 1. MT-SP1 was not detectable by RT-PCR in adjacent normal
tissue of either case.
[0379] Immunohistochemistry.
[0380] We used the immunohistochemical assay to study MT-SP1
expression in gastric cancer tissues. We detected MT-SP1
immunoreactivity in cancer cells, the luminal surface of blood
vessels, presumably endothelial cells, and some leukocytes. Since
the immunoreactivity in blood vessels in cancer stroma was
especially intense, we focused on the correlation between MT-SP1
expression in blood vessels and clinicopathologic factors in
gastric cancer.
[0381] Relationship Between MT-SP LI and Clinicopathologic
Factors.
[0382] MT-SP1 expression in blood vessels of cancer stroma did not
correlate with UICC TNM classification. Poorly differentiated
tumors showed significantly higher MT-SP LI than well
differentiated tumors. Tumors with pT1 showed low MT-SP LI as
compared to those with deeper invasion than submucosa but there
were no statistically significant differences.
[0383] Relationship Between MT-SP LI and Survival.
[0384] There were no significant differences in the overall
survival rates between the MT-SP positive group (MT-SP LI>0%)
and the MT-SP negative group (MT-SP LI=0%). However, when the
patients in the MT-SP positive group were divided into two groups
according to the average index (40%) in MT-SP LI as a cutoff point,
eleven patients with a high expression of MT-SP1 (MT-SP LI>=40%)
in blood vessels showed a lower survival rate than 21 patients with
low expression of MT-SP1 (MT-SP LI: 40%) or 9 patients with no
expression of MT-SP1 (P,0.05). The five-year survival rate for
patients with an MT-SP LI of 40% or higher was 27.3% versus 47.1%
for patients with an MT-SP LI lower than 40% or 45.7% for patients
with negative expression of MT-SP1.
[0385] Prognostic Analysis of MT-SP1 in Endothelium According to
Pathological Staging.
[0386] Kaplan-Meier survival calculations were performed for stages
I, II and stages III, IV separately. In stages III, IV, patients
(n=30) with an MT-SP LI of 40% or higher had a significantly lower
survival rate than those (n=11) without MT-SP1 expression in
vascular cells of cancer tissues (p<0.05). For stages I, II,
however, there was no difference in survival among the three
groups.
[0387] For multivariate analysis, MT-SP1 expression in blood
vessels around cancer cells and established risk factors in gastric
cancer were categorized into two classes. MT-SP1 expression was
subdivided into two categories; i) with an MT-SP LI less than 40%,
ii) with an MT-SP LI of 40% or higher. The depth of tumor invasion
and lymph node involvement were subdivided into two categories;
pT1,2 (n=19) and pT3,4 (n=22), pN0,1 (n=27) and pN2 (n=14),
respectively. These characteristics were confirmed to be
significant prognostic determinants in the generalized Wilcoxon
test. In Coxis regression model, higher expression of MT-SP1 in
blood vessels around cancer cells proved to worsen survival
independently, following serosal or deeper invasion of primary
tumor.
[0388] Discussion
[0389] MT-SP1 was identified, initially, in a prostate cancer cell
line. We believe it is involved in homeostatic processes occurring
in the pericellular milieu. The cDNA sequence of MT-SP1 encodes a
mosaic protein that contains a transmembrane signal anchor and a
serine protease domain, which is also seen in other membrane-type
serine proteases hepsine (Leytus et al. (1988) Biochemistry 27:
1067-1074), enteropeptidase (Kitamoto et al. (1994) Proc Natl Acad
Sci USA, 91: 7588-7592), and TMPRSS2 (Paoloni-Giacobino et al.
(1997) Genomics, 44: 309-320). Recently a similar cDNA was cloned
from mouse thymic stromal cells which was named epithin (Kim et al.
(1999) Immunogenetics 49: 420-428). An open reading frame was
identified that encoded a 902 amino acid protein with a C-terminal
serine protease domain, 4 LDLR domains, and two cub domains. A high
level of expression was seen by Northern blotting in mouse
intestine and kidney. mRNA was not detected in brain, heart, liver,
testis or skeletal muscle. In some tissues, different forms of mRNA
were seen suggesting the possibility of alternative splicing or
alternative polyadenylation sites. Epithin was localized to mouse
chromosome 9, .about.16 cM from the centromere.
[0390] It is thought that MT-SP1 which is localized to the plasma
membrane through a signal anchor is a component of proteolytic
cascades involved in developmental processes, and physiologic
reactions on the surface of the gastrointestinal and genito-urinary
epithelium. MT-SP1 is expressed in various malignant epithelial
tissues including the digestive and urinary tracts suggesting that
it may also have a role in cancer progression. Recently, Lin et al.
reported expression of a similar protease in human breast cancer
cells which was termed, Matriptase (Lin et al. (19_) J. Biol. Chem.
274: 18237-18242). A complex of this protease with a Kunitz-type
inhibitor was also fond in human breast milk (however, since the
characterization of its physiological substrate(s) have not been
completed, the function of this novel enzyme in cancer progression
is unclear).
[0391] In this example, we have clarified a relationship between
the expression of MT-SP1 and clinicopathological factors in gastric
cancer. The molecular weight of the entire MT-SP1 protein is
predicted as approximately 100 kD which is consistent with our
immunoblotting analysis. In this analysis, another band was shown
at the molecular weight of 45 kD. This band is likely to represent
the activated form of the protease domain of MT-SP1 that is
released from the prodomain under reducing conditions. Although the
expected molecular weight of this form is 28 kD, this difference
might be due to glycosylation of the protease domain.
[0392] Immunohistochemical examination of gastric cancer tissue
revealed MT-SP1 expression in cancer cells, endothelial cells and
some leukocytes. In these tissues, endothelial cells showed
especially intensive MT-SP1 immunoreactivity. This suggests that
MT-SP1 plays an important role in vascular cells. Although there
was not a significant correlation between MT-SP1 expression in
endothelial cells of gastric cancer tissue and pathological
staging, MT-SP1 was highly expressed in the endothelium of the
poorly differentiated adenocarcinomas. Moreover, overall survival
for groups of gastric carcinoma patients with highly MT-SP1
expressing endothelium revealed poor prognosis compared to those
with low or no MT-SP1. Higher MT-SP1 expression in endothelium was
significantly associated with lower survival rate. These results
suggested that MT-SP1 expression in endothelium around cancer cells
might be an important prognostic factor in gastric cancer.
[0393] MT-SP1 expression in vessels within the cancer matrix may
contribute to angiogenesis in gastric cancer tissue. Some
angiogenic factors such as vascular endothelial growth factor
(VEGF) (Ferrara-et al. (1991) Meth. Enzymol. 198: 391-405, Melnyk
(1996) Cancer Res. 56: 921-924) derived from cancer cells might be
associated with the MT-SP1 expression in endothelial cells. The
interaction between cancer cells and stromal cells in cancer tissue
is likely to be important for invasion and metastasis as described
in other reports (Romer et al. (1994) Int J. Cancer. 57: 553-560;
Sieuwerts et al. (1988) Int J. Cancer. 76: 829-835).
[0394] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
98 1 3142 DNA Homo sapiens CDS (37)..(2601) 1 cgaggatcct gagacccgcg
agcggcctcg gggacc atg ggg agc gat cgg gcc 54 Met Gly Ser Asp Arg
Ala 1 5 cgc aag ggc gga ggg ggc ccg aag gac ttc ggc gcg gga ctc aag
tac 102 Arg Lys Gly Gly Gly Gly Pro Lys Asp Phe Gly Ala Gly Leu Lys
Tyr 10 15 20 aac tcc cgg cac gag aaa gtg aat ggc ttg gag gaa ggc
gtg gag ttc 150 Asn Ser Arg His Glu Lys Val Asn Gly Leu Glu Glu Gly
Val Glu Phe 25 30 35 ctg cca gtc aac aac gtc aag aag gtg gaa aag
cat ggc ccg ggg cgc 198 Leu Pro Val Asn Asn Val Lys Lys Val Glu Lys
His Gly Pro Gly Arg 40 45 50 tgg gtg gtg ctg gca gcc gtg ctg atc
ggc ctc ctc ttg gtc ttg ctg 246 Trp Val Val Leu Ala Ala Val Leu Ile
Gly Leu Leu Leu Val Leu Leu 55 60 65 70 ggg atc ggc ttc ctg gtg tgg
cat ttg cag tac cgg gac gtg cgt gtc 294 Gly Ile Gly Phe Leu Val Trp
His Leu Gln Tyr Arg Asp Val Arg Val 75 80 85 cag aag gtc ttc aat
ggc tac atg agg atc aca aat gag aat ttt gtg 342 Gln Lys Val Phe Asn
Gly Tyr Met Arg Ile Thr Asn Glu Asn Phe Val 90 95 100 gat gcc tac
gag aac tcc aac tcc act gag ttt gta agc ctg gcc agc 390 Asp Ala Tyr
Glu Asn Ser Asn Ser Thr Glu Phe Val Ser Leu Ala Ser 105 110 115 aag
gtg aag gac gcg ctg aag ctg ctg tac agc gga gtc cca ttc ctg 438 Lys
Val Lys Asp Ala Leu Lys Leu Leu Tyr Ser Gly Val Pro Phe Leu 120 125
130 ggc ccc tac cac aag gag tcg gct gtg acg gcc ttc agc gag ggc agc
486 Gly Pro Tyr His Lys Glu Ser Ala Val Thr Ala Phe Ser Glu Gly Ser
135 140 145 150 gtc atc gcc tac tac tgg tct gag ttc agc atc ccg cag
cac ctg gtg 534 Val Ile Ala Tyr Tyr Trp Ser Glu Phe Ser Ile Pro Gln
His Leu Val 155 160 165 gag gag gcc gag cgc gtc atg gcc gag gag cgc
gta gtc atg ctg ccc 582 Glu Glu Ala Glu Arg Val Met Ala Glu Glu Arg
Val Val Met Leu Pro 170 175 180 ccg cgg gcg cgc tcc ctg aag tcc ttt
gtg gtc acc tca gtg gtg gct 630 Pro Arg Ala Arg Ser Leu Lys Ser Phe
Val Val Thr Ser Val Val Ala 185 190 195 ttc ccc acg gac tcc aaa aca
gta cag agg acc cag gac aac agc tgc 678 Phe Pro Thr Asp Ser Lys Thr
Val Gln Arg Thr Gln Asp Asn Ser Cys 200 205 210 agc ttt ggc ctg cac
gcc cgc ggt gtg gag ctg atg cgc ttc acc acg 726 Ser Phe Gly Leu His
Ala Arg Gly Val Glu Leu Met Arg Phe Thr Thr 215 220 225 230 ccc ggc
ttc cct gac agc ccc tac ccc gct cat gcc cgc tgc cag tgg 774 Pro Gly
Phe Pro Asp Ser Pro Tyr Pro Ala His Ala Arg Cys Gln Trp 235 240 245
gcc ctg cgg ggg gac gcc gac tca gtg ctg agc ctc acc ttc cgc agc 822
Ala Leu Arg Gly Asp Ala Asp Ser Val Leu Ser Leu Thr Phe Arg Ser 250
255 260 ttt gac ctt gcg tcc tgc gac gag cgc ggc agc gac ctg gtg acg
gtg 870 Phe Asp Leu Ala Ser Cys Asp Glu Arg Gly Ser Asp Leu Val Thr
Val 265 270 275 tac aac acc ctg agc ccc atg gag ccc cac gcc ctg gtg
cag ttg tgt 918 Tyr Asn Thr Leu Ser Pro Met Glu Pro His Ala Leu Val
Gln Leu Cys 280 285 290 ggc acc tac cct ccc tcc tac aac ctg acc ttc
cac tcc tcc cag aac 966 Gly Thr Tyr Pro Pro Ser Tyr Asn Leu Thr Phe
His Ser Ser Gln Asn 295 300 305 310 gtc ctg ctc atc aca ctg ata acc
aac act gag cgg cgg cat ccc ggc 1014 Val Leu Leu Ile Thr Leu Ile
Thr Asn Thr Glu Arg Arg His Pro Gly 315 320 325 ttt gag gcc acc ttc
ttc cag ctg cct agg atg agc agc tgt gga ggc 1062 Phe Glu Ala Thr
Phe Phe Gln Leu Pro Arg Met Ser Ser Cys Gly Gly 330 335 340 cgc tta
cgt aaa gcc cag ggg aca ttc aac agc ccc tac tac cca ggc 1110 Arg
Leu Arg Lys Ala Gln Gly Thr Phe Asn Ser Pro Tyr Tyr Pro Gly 345 350
355 cac tac cca ccc aac att gac tgc aca tgg aac att gag gtg ccc aac
1158 His Tyr Pro Pro Asn Ile Asp Cys Thr Trp Asn Ile Glu Val Pro
Asn 360 365 370 aac cag cat gtg aag gtg cgc ttc aaa ttc ttc tac ctg
ctg gag ccc 1206 Asn Gln His Val Lys Val Arg Phe Lys Phe Phe Tyr
Leu Leu Glu Pro 375 380 385 390 ggc gtg cct gcg ggc acc tgc ccc aag
gac tac gtg gag atc aat ggg 1254 Gly Val Pro Ala Gly Thr Cys Pro
Lys Asp Tyr Val Glu Ile Asn Gly 395 400 405 gag aaa tac tgc gga gag
agg tcc cag ttc gtc gtc acc agc aac agc 1302 Glu Lys Tyr Cys Gly
Glu Arg Ser Gln Phe Val Val Thr Ser Asn Ser 410 415 420 aac aag atc
aca gtt cgc ttc cac tca gat cag tcc tac acc gac acc 1350 Asn Lys
Ile Thr Val Arg Phe His Ser Asp Gln Ser Tyr Thr Asp Thr 425 430 435
ggc ttc tta gct gaa tac ctc tcc tac gac tcc agt gac cca tgc ccg
1398 Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp Ser Ser Asp Pro Cys
Pro 440 445 450 ggg cag ttc acg tgc cgc acg ggg cgg tgt atc cgg aag
gag ctg cgc 1446 Gly Gln Phe Thr Cys Arg Thr Gly Arg Cys Ile Arg
Lys Glu Leu Arg 455 460 465 470 tgt gat ggc tgg gcc gac tgc acc gac
cac agc gat gag ctc aac tgc 1494 Cys Asp Gly Trp Ala Asp Cys Thr
Asp His Ser Asp Glu Leu Asn Cys 475 480 485 agt tgc gac gcc ggc cac
cag ttc acg tgc aag aac aag ttc tgc aag 1542 Ser Cys Asp Ala Gly
His Gln Phe Thr Cys Lys Asn Lys Phe Cys Lys 490 495 500 ccc ctc ttc
tgg gtc tgc gac agt gtg aac gac tgc gga gac aac agc 1590 Pro Leu
Phe Trp Val Cys Asp Ser Val Asn Asp Cys Gly Asp Asn Ser 505 510 515
gac gag cag ggg tgc agt tgt ccg gcc cag acc ttc agg tgt tcc aat
1638 Asp Glu Gln Gly Cys Ser Cys Pro Ala Gln Thr Phe Arg Cys Ser
Asn 520 525 530 ggg aag tgc ctc tcg aaa agc cag cag tgc aat ggg aag
gac gac tgt 1686 Gly Lys Cys Leu Ser Lys Ser Gln Gln Cys Asn Gly
Lys Asp Asp Cys 535 540 545 550 ggg gac ggg tcc gac gag gcc tcc tgc
ccc aag gtg aac gtc gtc act 1734 Gly Asp Gly Ser Asp Glu Ala Ser
Cys Pro Lys Val Asn Val Val Thr 555 560 565 tgt acc aaa cac acc tac
cgc tgc ctc aat ggg ctc tgc ttg agc aag 1782 Cys Thr Lys His Thr
Tyr Arg Cys Leu Asn Gly Leu Cys Leu Ser Lys 570 575 580 ggc aac cct
gag tgt gac ggg aag gag gac tgt agc gac ggc tca gat 1830 Gly Asn
Pro Glu Cys Asp Gly Lys Glu Asp Cys Ser Asp Gly Ser Asp 585 590 595
gag aag gac tgc gac tgt ggg ctg cgg tca ttc acg aga cag gct cgt
1878 Glu Lys Asp Cys Asp Cys Gly Leu Arg Ser Phe Thr Arg Gln Ala
Arg 600 605 610 gtt gtt ggg ggc acg gat gcg gat gag ggc gag tgg ccc
tgg cag gta 1926 Val Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp
Pro Trp Gln Val 615 620 625 630 agc ctg cat gct ctg ggc cag ggc cac
atc tgc ggt gct tcc ctc atc 1974 Ser Leu His Ala Leu Gly Gln Gly
His Ile Cys Gly Ala Ser Leu Ile 635 640 645 tct ccc aac tgg ctg gtc
tct gcc gca cac tgc tac atc gat gac aga 2022 Ser Pro Asn Trp Leu
Val Ser Ala Ala His Cys Tyr Ile Asp Asp Arg 650 655 660 gga ttc agg
tac tca gac ccc acg cag tgg acg gcc ttc ctg ggc ttg 2070 Gly Phe
Arg Tyr Ser Asp Pro Thr Gln Trp Thr Ala Phe Leu Gly Leu 665 670 675
cac gac cag agc cag cgc agc gcc cct ggg gtg cag gag cgc agg ctc
2118 His Asp Gln Ser Gln Arg Ser Ala Pro Gly Val Gln Glu Arg Arg
Leu 680 685 690 aag cgc atc atc tcc cac ccc ttc ttc aat gac ttc acc
ttc gac tat 2166 Lys Arg Ile Ile Ser His Pro Phe Phe Asn Asp Phe
Thr Phe Asp Tyr 695 700 705 710 gac atc gcg ctg ctg gag ctg gag aaa
ccg gca gag tac agc tcc atg 2214 Asp Ile Ala Leu Leu Glu Leu Glu
Lys Pro Ala Glu Tyr Ser Ser Met 715 720 725 gtg cgg ccc atc tgc ctg
ccg gac gcc tcc cat gtc ttc cct gcc ggc 2262 Val Arg Pro Ile Cys
Leu Pro Asp Ala Ser His Val Phe Pro Ala Gly 730 735 740 aag gcc atc
tgg gtc acg ggc tgg gga cac acc cag tat gga ggc act 2310 Lys Ala
Ile Trp Val Thr Gly Trp Gly His Thr Gln Tyr Gly Gly Thr 745 750 755
ggc gcg ctg atc ctg caa aag ggt gag atc cgc gtc atc aac cag acc
2358 Gly Ala Leu Ile Leu Gln Lys Gly Glu Ile Arg Val Ile Asn Gln
Thr 760 765 770 acc tgc gag aac ctc ctg ccg cag cag atc acg ccg cgc
atg atg tgc 2406 Thr Cys Glu Asn Leu Leu Pro Gln Gln Ile Thr Pro
Arg Met Met Cys 775 780 785 790 gtg ggc ttc ctc agc ggc ggc gtg gac
tcc tgc cag ggt gat tcc ggg 2454 Val Gly Phe Leu Ser Gly Gly Val
Asp Ser Cys Gln Gly Asp Ser Gly 795 800 805 gga ccc ctg tcc agc gtg
gag gcg gat ggg cgg atc ttc cag gcc ggt 2502 Gly Pro Leu Ser Ser
Val Glu Ala Asp Gly Arg Ile Phe Gln Ala Gly 810 815 820 gtg gtg agc
tgg gga gac ggc tgc gct cag agg aac aag cca ggc gtg 2550 Val Val
Ser Trp Gly Asp Gly Cys Ala Gln Arg Asn Lys Pro Gly Val 825 830 835
tac aca agg ctc cct ctg ttt cgg gac tgg atc aaa gag aac act ggg
2598 Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp Ile Lys Glu Asn Thr
Gly 840 845 850 gta taggggccgg ggcacccaag atgtgtacac ctgcggggcc
acccatcgtc 2651 Val 855 caccccagtg tgcacgcctg caggctggag actggaccgc
tgactgcacc agcgccccca 2711 gaacatacac tgtgaactca atctccaggg
ctccaaatct gcctagaaaa cctctcgctt 2771 cctcagcctc caaagtggag
ctgggaggta gaaggggagg acactggtgg ttctactgac 2831 ccaactgggg
gcaaaggttt gaagacacag cctcccccgc cagccccaag ctgggccgag 2891
gcgcgtttgt gtatatctgc ctcccctgtc tgtaaggagc agcgggaacg gagcttcggg
2951 gcctcctcag tgaaggtggt ggggctgccg gatctgggct gtggggccct
tgggccacgc 3011 tcttgaggaa gcccaggctc ggaggaccct ggaaaacaga
cgggtctgag actgaaattg 3071 ttttaccagc tcccagggtg gacttcagtg
tgtgtatttg tgtaaatgag taaaacattt 3131 tatttctttt t 3142 2 855 PRT
Homo sapiens 2 Met Gly Ser Asp Arg Ala Arg Lys Gly Gly Gly Gly Pro
Lys Asp Phe 1 5 10 15 Gly Ala Gly Leu Lys Tyr Asn Ser Arg His Glu
Lys Val Asn Gly Leu 20 25 30 Glu Glu Gly Val Glu Phe Leu Pro Val
Asn Asn Val Lys Lys Val Glu 35 40 45 Lys His Gly Pro Gly Arg Trp
Val Val Leu Ala Ala Val Leu Ile Gly 50 55 60 Leu Leu Leu Val Leu
Leu Gly Ile Gly Phe Leu Val Trp His Leu Gln 65 70 75 80 Tyr Arg Asp
Val Arg Val Gln Lys Val Phe Asn Gly Tyr Met Arg Ile 85 90 95 Thr
Asn Glu Asn Phe Val Asp Ala Tyr Glu Asn Ser Asn Ser Thr Glu 100 105
110 Phe Val Ser Leu Ala Ser Lys Val Lys Asp Ala Leu Lys Leu Leu Tyr
115 120 125 Ser Gly Val Pro Phe Leu Gly Pro Tyr His Lys Glu Ser Ala
Val Thr 130 135 140 Ala Phe Ser Glu Gly Ser Val Ile Ala Tyr Tyr Trp
Ser Glu Phe Ser 145 150 155 160 Ile Pro Gln His Leu Val Glu Glu Ala
Glu Arg Val Met Ala Glu Glu 165 170 175 Arg Val Val Met Leu Pro Pro
Arg Ala Arg Ser Leu Lys Ser Phe Val 180 185 190 Val Thr Ser Val Val
Ala Phe Pro Thr Asp Ser Lys Thr Val Gln Arg 195 200 205 Thr Gln Asp
Asn Ser Cys Ser Phe Gly Leu His Ala Arg Gly Val Glu 210 215 220 Leu
Met Arg Phe Thr Thr Pro Gly Phe Pro Asp Ser Pro Tyr Pro Ala 225 230
235 240 His Ala Arg Cys Gln Trp Ala Leu Arg Gly Asp Ala Asp Ser Val
Leu 245 250 255 Ser Leu Thr Phe Arg Ser Phe Asp Leu Ala Ser Cys Asp
Glu Arg Gly 260 265 270 Ser Asp Leu Val Thr Val Tyr Asn Thr Leu Ser
Pro Met Glu Pro His 275 280 285 Ala Leu Val Gln Leu Cys Gly Thr Tyr
Pro Pro Ser Tyr Asn Leu Thr 290 295 300 Phe His Ser Ser Gln Asn Val
Leu Leu Ile Thr Leu Ile Thr Asn Thr 305 310 315 320 Glu Arg Arg His
Pro Gly Phe Glu Ala Thr Phe Phe Gln Leu Pro Arg 325 330 335 Met Ser
Ser Cys Gly Gly Arg Leu Arg Lys Ala Gln Gly Thr Phe Asn 340 345 350
Ser Pro Tyr Tyr Pro Gly His Tyr Pro Pro Asn Ile Asp Cys Thr Trp 355
360 365 Asn Ile Glu Val Pro Asn Asn Gln His Val Lys Val Arg Phe Lys
Phe 370 375 380 Phe Tyr Leu Leu Glu Pro Gly Val Pro Ala Gly Thr Cys
Pro Lys Asp 385 390 395 400 Tyr Val Glu Ile Asn Gly Glu Lys Tyr Cys
Gly Glu Arg Ser Gln Phe 405 410 415 Val Val Thr Ser Asn Ser Asn Lys
Ile Thr Val Arg Phe His Ser Asp 420 425 430 Gln Ser Tyr Thr Asp Thr
Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp 435 440 445 Ser Ser Asp Pro
Cys Pro Gly Gln Phe Thr Cys Arg Thr Gly Arg Cys 450 455 460 Ile Arg
Lys Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Thr Asp His 465 470 475
480 Ser Asp Glu Leu Asn Cys Ser Cys Asp Ala Gly His Gln Phe Thr Cys
485 490 495 Lys Asn Lys Phe Cys Lys Pro Leu Phe Trp Val Cys Asp Ser
Val Asn 500 505 510 Asp Cys Gly Asp Asn Ser Asp Glu Gln Gly Cys Ser
Cys Pro Ala Gln 515 520 525 Thr Phe Arg Cys Ser Asn Gly Lys Cys Leu
Ser Lys Ser Gln Gln Cys 530 535 540 Asn Gly Lys Asp Asp Cys Gly Asp
Gly Ser Asp Glu Ala Ser Cys Pro 545 550 555 560 Lys Val Asn Val Val
Thr Cys Thr Lys His Thr Tyr Arg Cys Leu Asn 565 570 575 Gly Leu Cys
Leu Ser Lys Gly Asn Pro Glu Cys Asp Gly Lys Glu Asp 580 585 590 Cys
Ser Asp Gly Ser Asp Glu Lys Asp Cys Asp Cys Gly Leu Arg Ser 595 600
605 Phe Thr Arg Gln Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly
610 615 620 Glu Trp Pro Trp Gln Val Ser Leu His Ala Leu Gly Gln Gly
His Ile 625 630 635 640 Cys Gly Ala Ser Leu Ile Ser Pro Asn Trp Leu
Val Ser Ala Ala His 645 650 655 Cys Tyr Ile Asp Asp Arg Gly Phe Arg
Tyr Ser Asp Pro Thr Gln Trp 660 665 670 Thr Ala Phe Leu Gly Leu His
Asp Gln Ser Gln Arg Ser Ala Pro Gly 675 680 685 Val Gln Glu Arg Arg
Leu Lys Arg Ile Ile Ser His Pro Phe Phe Asn 690 695 700 Asp Phe Thr
Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro 705 710 715 720
Ala Glu Tyr Ser Ser Met Val Arg Pro Ile Cys Leu Pro Asp Ala Ser 725
730 735 His Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp Gly
His 740 745 750 Thr Gln Tyr Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys
Gly Glu Ile 755 760 765 Arg Val Ile Asn Gln Thr Thr Cys Glu Asn Leu
Leu Pro Gln Gln Ile 770 775 780 Thr Pro Arg Met Met Cys Val Gly Phe
Leu Ser Gly Gly Val Asp Ser 785 790 795 800 Cys Gln Gly Asp Ser Gly
Gly Pro Leu Ser Ser Val Glu Ala Asp Gly 805 810 815 Arg Ile Phe Gln
Ala Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gln 820 825 830 Arg Asn
Lys Pro Gly Val Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp 835 840 845
Ile Lys Glu Asn Thr Gly Val 850 855 3 241 PRT Artificial Sequence
Description of Artificial Sequence Synthetic MT-SP1 fragment 3 Val
Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp Pro Trp Gln Val 1 5 10
15 Ser Leu His Ala Leu Gly Gln Gly His Ile Cys Gly Ala Ser Leu Ile
20 25 30 Ser Pro Asn Trp Leu Val Ser Ala Ala His Cys Tyr Ile Asp
Asp Arg 35 40 45 Gly Phe Arg Tyr Ser Asp Pro Thr Gln Trp Thr Ala
Phe Leu Gly Leu 50 55 60 His Asp Gln Ser Gln Arg Ser Ala Pro Gly
Val Gln Glu Arg Arg Leu 65 70 75 80 Lys
Arg Ile Ile Ser His Pro Phe Phe Asn Asp Phe Thr Phe Asp Tyr 85 90
95 Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro Ala Glu Tyr Ser Ser Met
100 105 110 Val Arg Pro Ile Cys Leu Pro Asp Ala Ser His Val Phe Pro
Ala Gly 115 120 125 Lys Ala Ile Trp Val Thr Gly Trp Gly His Thr Gln
Tyr Gly Gly Thr 130 135 140 Gly Ala Leu Ile Leu Gln Lys Gly Glu Ile
Arg Val Ile Asn Gln Thr 145 150 155 160 Thr Cys Glu Asn Leu Leu Pro
Gln Gln Ile Thr Pro Arg Met Met Cys 165 170 175 Val Gly Phe Leu Ser
Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly 180 185 190 Gly Pro Leu
Ser Ser Val Glu Ala Asp Gly Arg Ile Phe Gln Ala Gly 195 200 205 Val
Val Ser Trp Gly Asp Gly Cys Ala Gln Arg Asn Lys Pro Gly Val 210 215
220 Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp Ile Lys Glu Asn Thr Gly
225 230 235 240 Val 4 235 PRT Artificial Sequence Description of
Artificial Sequence Synthetic protein fragment/domain 4 Ile Val Gly
Gly Gln Glu Ala Pro Arg Ser Lys Trp Pro Trp Gln Val 1 5 10 15 Ser
Leu Arg Val His Asp Arg Tyr Trp Met His Phe Cys Gly Gly Ser 20 25
30 Leu Ile His Pro Gln Trp Val Leu Thr Ala Ala His Cys Val Gly Pro
35 40 45 Asp Val Lys Asp Leu Ala Ala Leu Arg Val Gln Leu Arg Glu
Gln His 50 55 60 Leu Tyr Tyr Gln Asp Gln Leu Leu Pro Val Ser Arg
Ile Ile Val His 65 70 75 80 Pro Gln Phe Tyr Thr Ala Gln Ile Gly Ala
Asp Ile Ala Leu Leu Glu 85 90 95 Leu Glu Glu Pro Val Lys Val Ser
Ser His Val His Thr Val Thr Leu 100 105 110 Pro Pro Ala Ser Glu Thr
Phe Pro Pro Gly Met Pro Cys Trp Val Thr 115 120 125 Gly Trp Gly Asp
Val Asp Asn Asp Glu Arg Leu Pro Pro Pro Phe Pro 130 135 140 Leu Lys
Gln Val Lys Val Pro Ile Met Glu Asn His Ile Cys Asp Ala 145 150 155
160 Lys Tyr His Leu Gly Ala Tyr Thr Gly Asp Asp Val Arg Ile Val Arg
165 170 175 Asp Asp Met Leu Cys Ala Gly Asn Thr Arg Arg Asp Ser Cys
Gln Gly 180 185 190 Asp Ser Gly Gly Pro Leu Val Cys Lys Val Asn Gly
Thr Trp Leu Gln 195 200 205 Ala Gly Val Val Ser Trp Gly Glu Gly Cys
Ala Gln Pro Asn Arg Pro 210 215 220 Gly Ile Tyr Thr Arg Val Val Pro
Lys Lys Pro 225 230 235 5 235 PRT Artificial Sequence Description
of Artificial Sequence Synthetic protein fragment/domain 5 Ile Val
Gly Gly Ser Asn Ala Lys Glu Gly Ala Trp Pro Trp Val Val 1 5 10 15
Gly Leu Tyr Tyr Gly Gly Arg Leu Leu Cys Gly Ala Ser Leu Val Ser 20
25 30 Ser Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn
Leu 35 40 45 Glu Pro Ser Lys Trp Thr Ala Ile Leu Gly Leu His Met
Lys Ser Asn 50 55 60 Leu Thr Ser Pro Gln Thr Val Pro Arg Leu Ile
Asp Glu Ile Val Ile 65 70 75 80 Asn Pro His Tyr Asn Arg Arg Arg Lys
Asp Asn Asp Ile Ala Met Met 85 90 95 His Leu Glu Phe Lys Val Asn
Tyr Thr Asp Tyr Ile Gln Pro Ile Cys 100 105 110 Leu Pro Glu Glu Asn
Gln Val Phe Pro Pro Gly Arg Asn Cys Ser Ile 115 120 125 Ala Gly Trp
Gly Thr Val Val Tyr Gln Gly Thr Thr Ala Asn Ile Leu 130 135 140 Gln
Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln 145 150
155 160 Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr
Glu 165 170 175 Glu Gly Gly Ile Asp Ser Cys Gln Gly Asp Ser Gly Gly
Pro Leu Met 180 185 190 Cys Gln Glu Asn Asn Arg Trp Phe Leu Ala Gly
Val Thr Ser Phe Gly 195 200 205 Tyr Lys Cys Ala Leu Pro Asn Arg Pro
Gly Val Tyr Ala Arg Val Ser 210 215 220 Arg Phe Thr Glu Trp Ile Gln
Ser Phe Leu His 225 230 235 6 255 PRT Artificial Sequence
Description of Artificial Sequence Synthetic protein
fragment/domain 6 Ile Val Gly Gly Arg Asp Thr Ser Leu Gly Arg Trp
Pro Trp Gln Val 1 5 10 15 Ser Leu Arg Tyr Asp Gly Ala His Leu Cys
Gly Gly Ser Leu Leu Ser 20 25 30 Gly Asp Trp Val Leu Thr Ala Ala
His Cys Phe Pro Glu Arg Asn Arg 35 40 45 Val Leu Ser Arg Trp Arg
Val Phe Ala Gly Ala Val Ala Gln Ala Ser 50 55 60 Pro His Gly Leu
Gln Leu Gly Val Gln Ala Val Val Tyr His Gly Gly 65 70 75 80 Tyr Leu
Pro Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser Asn Asp Ile 85 90 95
Ala Leu Val His Leu Ser Ser Pro Leu Pro Leu Thr Glu Tyr Ile Gln 100
105 110 Pro Val Cys Leu Pro Ala Ala Gly Gln Ala Leu Val Asp Gly Lys
Ile 115 120 125 Cys Thr Val Thr Gly Trp Gly Asn Thr Gln Tyr Tyr Gly
Gln Gln Ala 130 135 140 Gly Val Leu Gln Glu Ala Arg Val Pro Ile Ile
Ser Asn Asp Val Cys 145 150 155 160 Asn Gly Ala Asp Phe Tyr Gly Asn
Gln Ile Lys Pro Lys Met Phe Cys 165 170 175 Ala Gly Tyr Pro Glu Gly
Gly Ile Asp Ala Cys Gln Gly Asp Ser Gly 180 185 190 Gly Pro Phe Val
Cys Glu Asp Ser Ile Ser Arg Thr Pro Arg Trp Arg 195 200 205 Leu Cys
Gly Ile Val Ser Trp Gly Thr Gly Cys Ala Leu Ala Gln Lys 210 215 220
Pro Gly Val Tyr Thr Lys Val Ser Asp Phe Arg Glu Trp Ile Phe Gln 225
230 235 240 Ala Ile Lys Thr His Ser Glu Ala Ser Gly Met Val Thr Gln
Leu 245 250 255 7 224 PRT Artificial Sequence Description of
Artificial Sequence Synthetic protein fragment/domain 7 Ile Val Gly
Gly Tyr Ile Cys Glu Glu Asn Ser Val Pro Tyr Gln Val 1 5 10 15 Ser
Leu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu Ile Ser Glu 20 25
30 Gln Trp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45 Arg Leu Gly Glu His Asn Ile Glu Val Leu Glu Gly Asn Glu
Gln Phe 50 55 60 Ile Asn Ala Ala Lys Ile Ile Arg His Pro Lys Tyr
Asn Ser Arg Thr 65 70 75 80 Leu Asp Asn Asp Ile Leu Leu Ile Lys Leu
Ser Ser Pro Ala Val Ile 85 90 95 Asn Ser Arg Val Ser Ala Ile Ser
Leu Pro Thr Ala Pro Pro Ala Ala 100 105 110 Gly Thr Glu Ser Leu Ile
Ser Gly Trp Gly Asn Thr Leu Ser Ser Gly 115 120 125 Ala Asp Tyr Pro
Asp Glu Leu Gln Cys Leu Asp Ala Pro Val Leu Ser 130 135 140 Gln Ala
Glu Cys Glu Ala Ser Tyr Pro Gly Lys Ile Thr Asn Asn Met 145 150 155
160 Phe Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175 Ser Gly Gly Pro Val Val Ser Asn Gly Glu Leu Gln Gly Ile
Val Ser 180 185 190 Trp Gly Tyr Gly Cys Ala Gln Lys Asn Arg Pro Gly
Val Tyr Thr Lys 195 200 205 Val Tyr Asn Tyr Val Asp Trp Ile Lys Asp
Thr Ile Ala Ala Asn Ser 210 215 220 8 230 PRT Artificial Sequence
Description of Artificial Sequence Synthetic protein
fragment/domain 8 Ile Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp
Pro Trp Gln Val 1 5 10 15 Ser Leu Gln Asp Lys Thr Gly Phe His Phe
Cys Gly Gly Ser Leu Ile 20 25 30 Ser Glu Asp Trp Val Val Thr Ala
Ala His Cys Gly Val Arg Thr Ser 35 40 45 Asp Val Val Val Ala Gly
Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn 50 55 60 Ile Gln Val Leu
Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser 65 70 75 80 Ile Leu
Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro 85 90 95
Ala Arg Phe Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp 100
105 110 Asp Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly
Lys 115 120 125 Thr Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln
Gln Ala Ala 130 135 140 Leu Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys
Ser Trp Gly Arg Arg 145 150 155 160 Ile Thr Asp Val Met Ile Cys Ala
Gly Ala Ser Gly Val Ser Ser Cys 165 170 175 Met Gly Asp Ser Gly Gly
Pro Leu Val Cys Gln Lys Asp Gly Ala Trp 180 185 190 Thr Leu Val Gly
Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser 195 200 205 Ser Pro
Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln 210 215 220
Lys Ile Leu Ala Ala Asn 225 230 9 274 PRT Artificial Sequence
Description of Artificial Sequence Synthetic consensussequence
MOD_RES (5)..(9) Variable amino acid MOD_RES (11) Variable amino
acid MOD_RES (19)..(26) Variable amino acid MOD_RES (28) Variable
amino acid MOD_RES (36)..(37) Variable amino acid MOD_RES
(46)..(59) Variable amino acid MOD_RES (61)..(63) Variable amino
acid MOD_RES (66)..(68) Variable amino acid MOD_RES (70)..(84)
Variable amino acid MOD_RES (87) Variable amino acid MOD_RES
(90)..(98) Variable amino acid MOD_RES (101)..(102) Variable amino
acid MOD_RES (104) Variable amino acid MOD_RES (108)..(110)
Variable amino acid MOD_RES (112)..(120) Variable amino acid
MOD_RES (122) Variable amino acid MOD_RES (130) Variable amino acid
MOD_RES (133) Variable amino acid MOD_RES (135)..(140) Variable
amino acid MOD_RES (142) Variable amino acid MOD_RES (148) Variable
amino acid MOD_RES (150)..(152) Variable amino acid MOD_RES
(157)..(158) Variable amino acid MOD_RES (160) Variable amino acid
MOD_RES (166) Variable amino acid MOD_RES (168) Variable amino acid
MOD_RES (170)..(171) Variable amino acid MOD_RES (173)..(175)
Variable amino acid MOD_RES (177)..(178) Variable amino acid
MOD_RES (181)..(184) Variable amino acid MOD_RES (186) Variable
amino acid MOD_RES (190)..(191) Variable amino acid MOD_RES
(193)..(200) Variable amino acid MOD_RES (203) Variable amino acid
MOD_RES (216)..(229) Variable amino acid MOD_RES (236) Variable
amino acid MOD_RES (241) Variable amino acid MOD_RES (251)..(252)
Variable amino acid MOD_RES (254) Variable amino acid MOD_RES
(258)..(274) Variable amino acid 9 Ile Val Gly Gly Xaa Xaa Xaa Xaa
Xaa Gly Xaa Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa His Xaa Cys Gly Gly Ser 20 25 30 Leu Ile Ser Xaa
Xaa Trp Val Val Ser Ala Ala His Cys Xaa Xaa Xaa 35 40 45 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Leu 50 55 60
Gly Xaa Xaa Xaa Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Ile Ile Xaa His Pro Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Ile Thr Xaa Xaa Met Xaa Cys Ala Gly Xaa
Xaa Xaa Tyr Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa
Asp Asn Asp Ile Ala Leu 115 120 125 Leu Xaa Leu Glu Xaa Pro Xaa Xaa
Xaa Xaa Xaa Xaa Val Xaa Pro Ile 130 135 140 Cys Leu Pro Xaa Ala Xaa
Xaa Xaa Phe Pro Ala Gly Xaa Xaa Cys Xaa 145 150 155 160 Val Thr Gly
Trp Gly Xaa Thr Xaa Tyr Xaa Xaa Gly Xaa Xaa Xaa Ala 165 170 175 Xaa
Xaa Leu Gln Xaa Xaa Xaa Xaa Pro Xaa Ile Ser Asn Xaa Xaa Cys 180 185
190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Gly Xaa Asp Ser Cys Gln Gly
195 200 205 Asp Ser Gly Gly Pro Leu Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Gly Val Val Ser Trp Gly Xaa
Gly Cys Ala Gln 225 230 235 240 Xaa Asn Arg Pro Gly Val Tyr Thr Arg
Val Xaa Xaa Phe Xaa Asp Trp 245 250 255 Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa Xaa 10 35 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
protein fragment/domain 10 Cys Pro Gly Gln Phe Thr Cys Arg Thr Gly
Arg Cys Ile Arg Lys Glu 1 5 10 15 Leu Arg Cys Asp Gly Trp Ala Asp
Cys Thr Asp His Ser Asp Glu Leu 20 25 30 Asn Cys Ser 35 11 37 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
protein fragment/domain 11 Cys Asp Ala Gly His Gln Phe Thr Cys Lys
Asn Lys Phe Cys Lys Pro 1 5 10 15 Leu Phe Trp Val Cys Asp Ser Val
Asn Asp Cys Gly Asp Asn Ser Asp 20 25 30 Glu Gln Gly Cys Ser 35 12
42 PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 12 Cys Pro Ala Gln Thr Phe Arg
Cys Ser Asn Gly Lys Cys Leu Ser Lys 1 5 10 15 Ser Gln Gln Cys Asn
Gly Lys Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ala Ser Cys
Pro Lys Val Asn Val Val Thr 35 40 13 37 PRT Artificial Sequence
Description of Artificial Sequence Synthetic protein
fragment/domain 13 Cys Thr Lys His Thr Tyr Arg Cys Leu Asn Gly Leu
Cys Leu Ser Lys 1 5 10 15 Gly Asn Pro Glu Cys Asp Gly Lys Glu Asp
Cys Ser Asp Gly Ser Asp 20 25 30 Glu Lys Asp Cys Asp 35 14 41 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
protein fragment/domain 14 Cys Glu Arg Asn Glu Phe Gln Cys Gln Asp
Gly Lys Cys Ile Ser Tyr 1 5 10 15 Lys Trp Val Cys Asp Gly Ser Ala
Glu Cys Gln Asp Gly Ser Asp Glu 20 25 30 Ser Gln Glu Thr Cys Leu
Ser Val Thr 35 40 15 41 PRT Artificial Sequence Description of
Artificial Sequence Synthetic protein fragment/domain 15 Cys Lys
Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn Arg Cys Ile 1 5 10 15
Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp Asn Gly Ser 20
25 30 Asp Glu Gln Gly Cys Pro Pro Lys Thr 35 40 16 38 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
protein fragment/domain 16 Cys Ser Gln Asp Glu Phe Arg Cys His Asp
Gly Lys Cys Ile Ser Arg 1 5 10 15 Gln Phe Val Cys Asp Ser Arg Asp
Cys Leu Asp Gly Ser Asp Glu Ala 20 25 30 Ser Cys Pro Val Leu Thr 35
17 49 PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 17 Cys Gly Pro Ala Ser Phe Gln
Cys Asn Ser Ser Thr Cys Ile Pro Gln 1 5 10 15 Leu Trp Ala Cys Asp
Asn Asp Pro Asp Cys Glu Asp Gly Ser Asp Glu 20 25 30 Trp Pro Gln
Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly Asp Ser Ser 35 40 45 Pro 18
39 PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 18 Cys Ser Ala Phe Glu Phe His
Cys Leu Ser Gly Glu Cys Ile His Ser 1 5 10 15 Ser Trp Arg Cys Asp
Gly Gly Pro Asp Cys Lys Asp Lys Ser Asp Glu 20 25 30 Glu Asn Cys
Ala Val Ala Thr 35 19 40 PRT Artificial Sequence Description of
Artificial Sequence Synthetic protein fragment/domain 19 Cys Arg
Pro Asp Glu Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly 1 5 10 15
Ser Arg Gln Cys Asp Arg Glu Tyr Asp Cys Lys
Asp Met Ser Asp Glu 20 25 30 Val Gly Cys Val Asn Val Thr Leu 35 40
20 42 PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 20 Cys Glu Gly Pro Asn Lys Phe
Lys Cys His Ser Gly Glu Cys Ile Thr 1 5 10 15 Leu Asp Lys Val Cys
Asn Met Ala Arg Asp Cys Arg Asp Trp Ser Asp 20 25 30 Glu Pro Ile
Lys Glu Cys Gly Thr Asn Glu 35 40 21 53 PRT Artificial Sequence
Description of Artificial Sequence Synthetic consensussequence
MOD_RES (2)..(8) Variable amino acid MOD_RES (10)..(13) Variable
amino acid MOD_RES (15) Variable amino acid MOD_RES (18)..(21)
Variable amino acid MOD_RES (23) Variable amino acid MOD_RES
(27)..(28) Variable amino acid MOD_RES (31) Variable amino acid
MOD_RES (37)..(40) Variable amino acid MOD_RES (42)..(53) Variable
amino acid 21 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Gly Xaa Cys 1 5 10 15 Ile Xaa Xaa Xaa Xaa Trp Xaa Cys Asp Gly Xaa
Xaa Asp Cys Xaa Asp 20 25 30 Gly Ser Asp Glu Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa 50 22 126
PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 22 Cys Ser Phe Gly Leu His Ala
Arg Gly Val Glu Leu Met Arg Phe Thr 1 5 10 15 Thr Pro Gly Phe Pro
Asp Ser Pro Tyr Pro Ala His Ala Arg Cys Gln 20 25 30 Trp Ala Leu
Arg Gly Asp Ala Asp Ser Val Leu Ser Leu Thr Phe Arg 35 40 45 Ser
Phe Asp Leu Ala Ser Cys Asp Glu Arg Gly Ser Asp Leu Val Thr 50 55
60 Val Tyr Asn Thr Leu Ser Pro Met Glu Pro His Ala Leu Val Gln Leu
65 70 75 80 Cys Gly Thr Tyr Pro Pro Ser Tyr Asn Leu Thr Phe His Ser
Ser Gln 85 90 95 Asn Val Leu Leu Ile Thr Leu Ile Thr Asn Thr Glu
Arg Arg His Pro 100 105 110 Gly Phe Glu Ala Thr Phe Phe Gln Leu Pro
Arg Met Ser Ser 115 120 125 23 113 PRT Artificial Sequence
Description of Artificial Sequence Synthetic protein
fragment/domain 23 Cys Gly Gly Arg Leu Arg Lys Ala Gln Gly Thr Phe
Asn Ser Pro Tyr 1 5 10 15 Tyr Pro Gly His Tyr Pro Pro Asn Ile Asp
Cys Thr Trp Asn Ile Glu 20 25 30 Val Pro Asn Asn Gln His Val Lys
Val Arg Phe Lys Phe Phe Tyr Leu 35 40 45 Leu Glu Pro Gly Val Pro
Ala Gly Thr Cys Pro Lys Asp Tyr Val Glu 50 55 60 Ile Asn Gly Glu
Lys Tyr Cys Gly Glu Arg Ser Gln Phe Val Val Thr 65 70 75 80 Ser Asn
Ser Asn Lys Ile Thr Val Arg Phe His Ser Asp Gln Ser Tyr 85 90 95
Thr Asp Thr Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp Ser Ser Asp 100
105 110 Pro 24 110 PRT Artificial Sequence Description of
Artificial Sequence Synthetic protein fragment/domain 24 Cys Asp
Gly Arg Phe Leu Leu Thr Gly Ser Ser Gly Ser Phe Gln Ala 1 5 10 15
Thr His Tyr Pro Lys Pro Ser Glu Thr Ser Val Val Cys Gln Trp Ile 20
25 30 Ile Arg Val Asn Gln Gly Leu Ser Ile Lys Leu Ser Phe Asp Asp
Phe 35 40 45 Asn Thr Tyr Tyr Thr Asp Ile Leu Asp Ile Tyr Glu Gly
Val Gly Ser 50 55 60 Ser Lys Ile Leu Arg Ala Ser Ile Trp Glu Thr
Asn Pro Gly Thr Ile 65 70 75 80 Arg Ile Phe Ser Asn Gln Val Thr Ala
Thr Phe Leu Ile Glu Ser Asp 85 90 95 Glu Ser Asp Tyr Val Gly Phe
Asn Ala Thr Tyr Thr Ala Phe 100 105 110 25 111 PRT Artificial
Sequence Description of Artificial Sequence Synthetic protein
fragment/domain 25 Cys Gly Gly Pro Phe Glu Leu Trp Glu Pro Asn Thr
Thr Phe Ser Ser 1 5 10 15 Thr Asn Phe Pro Asn Ser Tyr Pro Asn Leu
Ala Phe Cys Val Trp Ile 20 25 30 Leu Asn Ala Gln Lys Gly Lys Asn
Ile Gln Leu His Phe Gln Glu Phe 35 40 45 Asp Leu Glu Asn Ile Asn
Asp Val Val Glu Ile Arg Asp Gly Glu Glu 50 55 60 Ala Asp Ser Leu
Leu Leu Ala Val Tyr Thr Gly Pro Gly Pro Val Lys 65 70 75 80 Asp Val
Phe Ser Thr Thr Asn Arg Met Thr Val Leu Leu Ile Thr Asn 85 90 95
Asp Val Leu Ala Arg Gly Gly Phe Lys Ala Asn Phe Thr Thr Gly 100 105
110 26 113 PRT Artificial Sequence Description of Artificial
Sequence Synthetic protein fragment/domain 26 Cys Gly Glu Thr Leu
Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro Glu 1 5 10 15 Tyr Pro Asn
Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile Ser 20 25 30 Val
Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp Leu 35 40
45 Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp Gly
50 55 60 Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser
Lys Leu 65 70 75 80 Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp
Val Glu Phe Arg 85 90 95 Ser Ser Ser Asn Trp Val Gly Lys Gly Phe
Phe Ala Val Tyr Glu Ala 100 105 110 Ile 27 112 PRT Artificial
Sequence Description of Artificial Sequence Synthetic protein
fragment/domain 27 Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile
Gln Ser Pro Asn 1 5 10 15 Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val
Cys Ile Trp Arg Ile Gln 20 25 30 Val Ser Glu Gly Phe His Val Gly
Leu Thr Phe Gln Ser Phe Glu Ile 35 40 45 Glu Arg His Asp Ser Cys
Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly 50 55 60 His Ser Glu Ser
Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr Glu Lys 65 70 75 80 Pro Asp
Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys Phe Val 85 90 95
Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe Lys 100
105 110 28 113 PRT Artificial Sequence Description of Artificial
Sequence Synthetic protein fragment/domain 28 Cys Gly Gly Phe Leu
Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly 1 5 10 15 Trp Pro Lys
Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val 20 25 30 Ala
Pro Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr 35 40
45 Glu Gly Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly
50 55 60 Leu Thr Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser
Glu Lys 65 70 75 80 Pro Glu Val Ile Thr Ser Gln Tyr Asn Asn Met Arg
Val Glu Phe Lys 85 90 95 Ser Asp Asn Thr Val Ser Lys Lys Gly Phe
Lys Ala His Phe Phe Ser 100 105 110 Glu 29 123 PRT Artificial
Sequence Description of Artificial Sequence Synthetic protein
fragment/domain 29 Ser Ile Pro Ile Pro Gln Lys Leu Phe Gly Glu Val
Thr Ser Pro Leu 1 5 10 15 Phe Pro Lys Pro Tyr Pro Asn Asn Phe Glu
Thr Thr Thr Val Ile Thr 20 25 30 Val Pro Thr Gly Tyr Arg Val Lys
Leu Val Phe Gln Gln Phe Asp Leu 35 40 45 Glu Pro Ser Glu Gly Cys
Phe Tyr Asp Tyr Val Lys Ile Ser Ala Asp 50 55 60 Lys Lys Leu Gly
Arg Phe Cys Gly Gln Leu Gly Ser Pro Leu Gly Asn 65 70 75 80 Pro Pro
Gly Lys Lys Glu Phe Met Ser Gln Gly Asn Lys Met Leu Leu 85 90 95
Thr Phe His Thr Asp Phe Ser Asn Glu Glu Asn Gly Thr Ile Met Phe 100
105 110 Tyr Lys Gly Phe Leu Ala Tyr Tyr Gln Ala Val 115 120 30 112
PRT Artificial Sequence Description of Artificial Sequence
Synthetic protein fragment/domain 30 Cys Ser Ser Glu Tyr Thr Glu
Ala Ser Gly Tyr Ile Ser Ser Leu Glu 1 5 10 15 Tyr Pro Arg Ser Tyr
Pro Pro Asp Leu Arg Cys Asn Tyr Ser Ile Arg 20 25 30 Val Glu Arg
Gly Leu Thr Leu His Leu Lys Phe Leu Glu Pro Phe Asp 35 40 45 Ile
Asp Asp His Gln Gln Val His Cys Pro Tyr Asp Gln Leu Gln Ile 50 55
60 Tyr Ala Asn Gly Lys Asn Ile Gly Glu Phe Cys Gly Lys Gln Arg Pro
65 70 75 80 Pro Asp Leu Asp Thr Ser Ser Asn Ala Val Asp Leu Leu Phe
Phe Thr 85 90 95 Asp Glu Ser Gly Asp Ser Arg Gly Trp Lys Leu Arg
Tyr Thr Thr Glu 100 105 110 31 143 PRT Artificial Sequence
Description of Artificial Sequence Synthetic consensussequence
MOD_RES (4) Variable amino acid MOD_RES (6)..(12) Variable amino
acid MOD_RES (14) Variable amino acid MOD_RES (19) Variable amino
acid MOD_RES (22)..(23) Variable amino acid MOD_RES (27)..(30)
Variable amino acid MOD_RES (32) Variable amino acid MOD_RES (34)
Variable amino acid MOD_RES (36) Variable amino acid MOD_RES
(38)..(39) Variable amino acid MOD_RES (41)..(42) Variable amino
acid MOD_RES (44) Variable amino acid MOD_RES (46) Variable amino
acid MOD_RES (48)..(50) Variable amino acid MOD_RES (54)..(62)
Variable amino acid MOD_RES (66)..(68) Variable amino acid MOD_RES
(75)..(76) Variable amino acid MOD_RES (78)..(85) Variable amino
acid MOD_RES (87)..(88) Variable amino acid MOD_RES (91)..(93)
Variable amino acid MOD_RES (95)..(96) Variable amino acid MOD_RES
(98)..(107) Variable amino acid MOD_RES (112)..(113) Variable amino
acid MOD_RES (115) Variable amino acid MOD_RES (117) Variable amino
acid MOD_RES (120) Variable amino acid MOD_RES (122)..(125)
Variable amino acid MOD_RES (127)..(129) Variable amino acid
MOD_RES (131) Variable amino acid MOD_RES (133) Variable amino acid
MOD_RES (135)..(143) Variable amino acid 31 Cys Gly Gly Xaa Leu Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Phe Ser 1 5 10 15 Ser Pro Xaa Tyr
Pro Xaa Xaa Tyr Pro Pro Xaa Xaa Xaa Xaa Cys Xaa 20 25 30 Trp Xaa
Ile Xaa Val Xaa Xaa Gly Xaa Xaa Ile Xaa Leu Xaa Phe Xaa 35 40 45
Xaa Xaa Phe Asp Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Pro 50
55 60 Tyr Xaa Xaa Xaa Asp Tyr Val Glu Ile Arg Xaa Xaa Gly Xaa Xaa
Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Cys Gly Xaa Xaa Xaa
Pro Xaa Xaa 85 90 95 Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ser Ser Asn Arg Xaa 100 105 110 Xaa Val Xaa Phe Xaa Ser Asp Xaa Ser
Xaa Xaa Xaa Xaa Gly Xaa Xaa 115 120 125 Xaa Phe Xaa Ala Xaa Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 32 30 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 32
tgcgacagtg tgaacgactg cggagacaac 30 33 30 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 33 ctccacgctg
gacaggggtc ccccggaatc 30 34 7 PRT Artificial Sequence Description
of Artificial Sequence Synthetic tag 34 Arg Gly Ser His His His His
1 5 35 241 PRT Homo sapiens 35 Val Val Gly Gly Thr Asp Ala Asp Glu
Gly Glu Trp Pro Trp Gln Val 1 5 10 15 Ser Leu His Ala Leu Gly Gln
Gly His Ile Cys Gly Ala Ser Leu Ile 20 25 30 Ser Pro Asn Trp Leu
Val Ser Ala Ala His Cys Tyr Ile Asp Asp Arg 35 40 45 Gly Phe Arg
Tyr Ser Asp Pro Thr Gln Trp Thr Ala Phe Leu Gly Leu 50 55 60 His
Asp Gln Ser Gln Arg Ser Ala Pro Gly Val Gln Glu Arg Arg Leu 65 70
75 80 Lys Arg Ile Ile Ser His Pro Phe Phe Asn Asp Phe Thr Phe Asp
Tyr 85 90 95 Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro Ala Glu Tyr
Ser Ser Met 100 105 110 Val Arg Pro Ile Cys Leu Pro Asp Ala Ser His
Val Phe Pro Ala Gly 115 120 125 Lys Ala Ile Trp Val Thr Gly Trp Gly
His Thr Gln Tyr Gly Gly Thr 130 135 140 Gly Ala Leu Ile Leu Gln Lys
Gly Glu Ile Arg Val Ile Asn Gln Thr 145 150 155 160 Thr Cys Glu Asn
Leu Leu Pro Gln Gln Ile Thr Pro Arg Met Met Cys 165 170 175 Val Gly
Phe Leu Ser Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly 180 185 190
Gly Pro Leu Ser Ser Val Glu Ala Asp Gly Arg Ile Phe Gln Ala Gly 195
200 205 Val Val Ser Trp Gly Asp Gly Cys Ala Gln Arg Asn Lys Pro Gly
Val 210 215 220 Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp Ile Lys Glu
Asn Thr Gly 225 230 235 240 Val 36 245 PRT Homo sapiens 36 Ile Val
Gly Gly Gln Glu Ala Pro Arg Ser Lys Trp Pro Trp Gln Val 1 5 10 15
Ser Leu Arg Val His Asp Arg Tyr Trp Met His Phe Cys Gly Gly Ser 20
25 30 Leu Ile His Pro Gln Trp Val Leu Thr Ala Ala His Cys Val Gly
Pro 35 40 45 Asp Val Lys Asp Leu Ala Ala Leu Arg Val Gln Leu Arg
Glu Gln His 50 55 60 Leu Tyr Tyr Gln Asp Gln Leu Leu Pro Val Ser
Arg Ile Ile Val His 65 70 75 80 Pro Gln Phe Tyr Thr Ala Gln Ile Gly
Ala Asp Ile Ala Leu Leu Glu 85 90 95 Leu Glu Glu Pro Val Lys Val
Ser Ser His Val His Thr Val Thr Leu 100 105 110 Pro Pro Ala Ser Glu
Thr Phe Pro Pro Gly Met Pro Cys Trp Val Thr 115 120 125 Gly Trp Gly
Asp Val Asp Asn Asp Glu Arg Leu Pro Pro Pro Phe Pro 130 135 140 Leu
Lys Gln Val Lys Val Pro Ile Met Glu Asn His Ile Cys Asp Ala 145 150
155 160 Lys Tyr His Leu Gly Ala Tyr Thr Gly Asp Asp Val Arg Ile Val
Arg 165 170 175 Asp Asp Met Leu Cys Ala Gly Asn Thr Arg Arg Asp Ser
Cys Gln Gly 180 185 190 Asp Ser Gly Gly Pro Leu Val Cys Lys Val Asn
Gly Thr Trp Leu Gln 195 200 205 Ala Gly Val Val Ser Trp Gly Glu Gly
Cys Ala Gln Pro Asn Arg Pro 210 215 220 Gly Ile Tyr Thr Arg Val Thr
Tyr Tyr Leu Asp Trp Ile His His Tyr 225 230 235 240 Val Pro Lys Lys
Pro 245 37 235 PRT Homo sapiens 37 Ile Val Gly Gly Ser Asn Ala Lys
Glu Gly Ala Trp Pro Trp Val Val 1 5 10 15 Gly Leu Tyr Tyr Gly Gly
Arg Leu Leu Cys Gly Ala Ser Leu Val Ser 20 25 30 Ser Asp Trp Leu
Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Leu 35 40 45 Glu Pro
Ser Lys Trp Thr Ala Ile Leu Gly Leu His Met Lys Ser Asn 50 55 60
Leu Thr Ser Pro Gln Thr Val Pro Arg Leu Ile Asp Glu Ile Val Ile 65
70 75 80 Asn Pro His Tyr Asn Arg Arg Arg Lys Asp Asn Asp Ile Ala
Met Met 85 90 95 His Leu Glu Phe Lys Val Asn Tyr Thr Asp Tyr Ile
Gln Pro Ile Cys 100 105 110 Leu Pro Glu Glu Asn Gln Val Phe Pro Pro
Gly Arg Asn Cys Ser Ile 115 120 125 Ala Gly Trp Gly Thr Val Val Tyr
Gln Gly Thr Thr Ala Asn Ile Leu 130 135 140 Gln Glu Ala Asp Val Pro
Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln 145 150 155 160 Met Pro Glu
Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu 165 170 175 Glu
Gly Gly Ile Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met 180 185
190 Cys Gln Glu Asn Asn Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly
195 200
205 Tyr Lys Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Ser
210 215 220 Arg Phe Thr Glu Trp Ile Gln Ser Phe Leu His 225 230 235
38 255 PRT Homo sapiens 38 Ile Val Gly Gly Arg Asp Thr Ser Leu Gly
Arg Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Arg Tyr Asp Gly Ala His
Leu Cys Gly Gly Ser Leu Leu Ser 20 25 30 Gly Asp Trp Val Leu Thr
Ala Ala His Cys Phe Pro Glu Arg Asn Arg 35 40 45 Val Leu Ser Arg
Trp Arg Val Phe Ala Gly Ala Val Ala Gln Ala Ser 50 55 60 Pro His
Gly Leu Gln Leu Gly Val Gln Ala Val Val Tyr His Gly Gly 65 70 75 80
Tyr Leu Pro Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser Asn Asp Ile 85
90 95 Ala Leu Val His Leu Ser Ser Pro Leu Pro Leu Thr Glu Tyr Ile
Gln 100 105 110 Pro Val Cys Leu Pro Ala Ala Gly Gln Ala Leu Val Asp
Gly Lys Ile 115 120 125 Cys Thr Val Thr Gly Trp Gly Asn Thr Gln Tyr
Tyr Gly Gln Gln Ala 130 135 140 Gly Val Leu Gln Glu Ala Arg Val Pro
Ile Ile Ser Asn Asp Val Cys 145 150 155 160 Asn Gly Ala Asp Phe Tyr
Gly Asn Gln Ile Lys Pro Lys Met Phe Cys 165 170 175 Ala Gly Tyr Pro
Glu Gly Gly Ile Asp Ala Cys Gln Gly Asp Ser Gly 180 185 190 Gly Pro
Phe Val Cys Glu Asp Ser Ile Ser Arg Thr Pro Arg Trp Arg 195 200 205
Leu Cys Gly Ile Val Ser Trp Gly Thr Gly Cys Ala Leu Ala Gln Lys 210
215 220 Pro Gly Val Tyr Thr Lys Val Ser Asp Phe Arg Glu Trp Ile Phe
Gln 225 230 235 240 Ala Ile Lys Thr His Ser Glu Ala Ser Gly Met Val
Thr Gln Leu 245 250 255 39 224 PRT Homo sapiens 39 Ile Val Gly Gly
Tyr Ile Cys Glu Glu Asn Ser Val Pro Tyr Gln Val 1 5 10 15 Ser Leu
Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu Ile Ser Glu 20 25 30
Gln Trp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Gln Val 35
40 45 Arg Leu Gly Glu His Asn Ile Glu Val Leu Glu Gly Asn Glu Gln
Phe 50 55 60 Ile Asn Ala Ala Lys Ile Ile Arg His Pro Lys Tyr Asn
Ser Arg Thr 65 70 75 80 Leu Asp Asn Asp Ile Leu Leu Ile Lys Leu Ser
Ser Pro Ala Val Ile 85 90 95 Asn Ser Arg Val Ser Ala Ile Ser Leu
Pro Thr Ala Pro Pro Ala Ala 100 105 110 Gly Thr Glu Ser Leu Ile Ser
Gly Trp Gly Asn Thr Leu Ser Ser Gly 115 120 125 Ala Asp Tyr Pro Asp
Glu Leu Gln Cys Leu Asp Ala Pro Val Leu Ser 130 135 140 Gln Ala Glu
Cys Glu Ala Ser Tyr Pro Gly Lys Ile Thr Asn Asn Met 145 150 155 160
Phe Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp 165
170 175 Ser Gly Gly Pro Val Val Ser Asn Gly Glu Leu Gln Gly Ile Val
Ser 180 185 190 Trp Gly Tyr Gly Cys Ala Gln Lys Asn Arg Pro Gly Val
Tyr Thr Lys 195 200 205 Val Tyr Asn Tyr Val Asp Trp Ile Lys Asp Thr
Ile Ala Ala Asn Ser 210 215 220 40 230 PRT Homo sapiens 40 Ile Val
Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val 1 5 10 15
Ser Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile 20
25 30 Ser Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr
Ser 35 40 45 Asp Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp
Glu Glu Asn 50 55 60 Ile Gln Val Leu Lys Ile Ala Lys Val Phe Lys
Asn Pro Lys Phe Ser 65 70 75 80 Ile Leu Thr Val Asn Asn Asp Ile Thr
Leu Leu Lys Leu Ala Thr Pro 85 90 95 Ala Arg Phe Ser Gln Thr Val
Ser Ala Val Cys Leu Pro Ser Ala Asp 100 105 110 Asp Asp Phe Pro Ala
Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys 115 120 125 Thr Lys Tyr
Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala 130 135 140 Leu
Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg 145 150
155 160 Ile Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser
Cys 165 170 175 Met Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp
Gly Ala Trp 180 185 190 Thr Leu Val Gly Ile Val Ser Trp Gly Ser Asp
Thr Cys Ser Thr Ser 195 200 205 Ser Pro Gly Val Tyr Ala Arg Val Thr
Lys Leu Ile Pro Trp Val Gln 210 215 220 Lys Ile Leu Ala Ala Asn 225
230 41 152 PRT Homo sapiens 41 Cys Pro Gly Gln Phe Thr Cys Arg Thr
Gly Arg Cys Ile Arg Lys Glu 1 5 10 15 Leu Arg Cys Asp Gly Trp Ala
Asp Cys Thr Asp His Ser Asp Glu Leu 20 25 30 Asn Cys Ser Cys Asp
Ala Gly His Gln Phe Thr Cys Lys Asn Lys Phe 35 40 45 Cys Lys Pro
Leu Phe Trp Val Cys Asp Ser Val Asn Asp Cys Gly Asp 50 55 60 Asn
Ser Asp Glu Gln Gly Cys Ser Cys Pro Ala Gln Thr Phe Arg Cys 65 70
75 80 Ser Asn Gly Lys Cys Leu Ser Lys Ser Gln Gln Cys Asn Gly Lys
Asp 85 90 95 Asp Cys Gly Asp Gly Ser Asp Glu Ala Ser Cys Pro Lys
Val Asn Val 100 105 110 Val Thr Cys Thr Lys His Thr Tyr Arg Cys Leu
Leu Asn Gly Leu Cys 115 120 125 Leu Ser Lys Gly Asn Pro Glu Cys Asp
Gly Lys Glu Asp Cys Ser Asp 130 135 140 Gly Ser Asp Glu Lys Asp Cys
Asp 145 150 42 290 PRT Homo sapiens 42 Cys Glu Arg Asn Glu Phe Gln
Cys Gln Asp Gly Lys Cys Ile Ser Tyr 1 5 10 15 Lys Trp Val Cys Asp
Gly Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu 20 25 30 Ser Gln Glu
Thr Cys Leu Ser Val Thr Cys Lys Ser Gly Asp Phe Ser 35 40 45 Cys
Gly Gly Arg Val Asn Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp 50 55
60 Gly Gln Val Asp Cys Asp Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro
65 70 75 80 Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His Asp Gly Lys
Cys Ile 85 90 95 Ser Arg Gln Phe Val Cys Asp Ser Arg Asp Cys Leu
Asp Gly Ser Asp 100 105 110 Glu Ala Ser Cys Pro Val Leu Thr Cys Gly
Pro Ala Ser Phe Gln Cys 115 120 125 Asn Ser Ser Thr Cys Ile Pro Gln
Leu Trp Ala Cys Asp Asn Asp Pro 130 135 140 Asp Cys Glu Asp Gly Ser
Asp Glu Trp Pro Gln Arg Cys Arg Gly Leu 145 150 155 160 Tyr Val Phe
Gln Gly Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His 165 170 175 Cys
Leu Ser Gly Glu Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly 180 185
190 Pro Asp Cys Lys Asp Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr
195 200 205 Cys Arg Pro Asp Glu Phe Gln Cys Ser Asp Gly Asn Cys Ile
His Gly 210 215 220 Ser Arg Gln Cys Asp Arg Glu Tyr Asp Cys Lys Asp
Met Ser Asp Glu 225 230 235 240 Val Gly Cys Val Asn Val Thr Leu Cys
Glu Gly Pro Asn Lys Phe Lys 245 250 255 Cys His Ser Gly Glu Cys Ile
Thr Leu Asp Lys Val Cys Asn Met Ala 260 265 270 Arg Asp Cys Arg Asp
Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr 275 280 285 Asn Glu 290
43 152 PRT Homo sapiens 43 Cys Ser Phe Gly Leu His Ala Arg Gly Val
Glu Leu Met Arg Phe Thr 1 5 10 15 Thr Pro Gly Phe Pro Asp Ser Pro
Tyr Pro Ala His Ala Arg Cys Gln 20 25 30 Trp Ala Leu Arg Gly Asp
Ala Asp Ser Val Leu Ser Leu Thr Phe Arg 35 40 45 Ser Phe Asp Leu
Ala Ser Cys Asp Glu Arg Gly Ser Asp Leu Val Thr 50 55 60 Val Tyr
Asn Thr Leu Ser Pro Met Glu Pro His Ala Leu Val Gln Leu 65 70 75 80
Cys Cys Gly Gly Arg Leu Arg Lys Ala Gln Gly Thr Phe Asn Ser Pro 85
90 95 Tyr Tyr Pro Gly His Tyr Pro Pro Asn Ile Asp Cys Thr Trp Asn
Ile 100 105 110 Glu Val Pro Asn Asn Gln His Val Lys Val Arg Phe Lys
Phe Phe Tyr 115 120 125 Leu Leu Glu Pro Gly Val Pro Ala Gly Thr Cys
Pro Lys Asp Tyr Val 130 135 140 Glu Ile Asn Gly Glu Lys Tyr Cys 145
150 44 74 PRT Homo sapiens 44 Cys Asp Gly Arg Phe Leu Leu Thr Gly
Ser Ser Gly Ser Phe Gln Ala 1 5 10 15 Thr His Tyr Pro Lys Pro Ser
Glu Thr Ser Val Val Cys Gln Trp Ile 20 25 30 Ile Arg Val Asn Gln
Gly Leu Ser Ile Lys Leu Ser Phe Asp Asp Phe 35 40 45 Asn Thr Tyr
Tyr Thr Asp Ile Leu Asp Ile Tyr Glu Gly Val Gly Ser 50 55 60 Ser
Lys Ile Leu Arg Ala Ser Ile Trp Glu 65 70 45 74 PRT Homo sapiens 45
Cys Gly Gly Pro Phe Glu Leu Trp Glu Pro Asn Thr Thr Phe Ser Ser 1 5
10 15 Thr Asn Phe Pro Asn Ser Tyr Pro Asn Leu Ala Phe Cys Val Trp
Ile 20 25 30 Leu Asn Ala Gln Lys Gly Lys Asn Ile Gln Leu His Phe
Gln Glu Phe 35 40 45 Asp Leu Glu Asn Ile Asn Asp Val Val Glu Ile
Arg Asp Gly Glu Glu 50 55 60 Ala Asp Ser Leu Leu Leu Ala Val Tyr
Thr 65 70 46 76 PRT Homo sapiens 46 Cys Gly Glu Thr Leu Gln Asp Ser
Thr Gly Asn Phe Ser Ser Pro Glu 1 5 10 15 Tyr Pro Asn Gly Tyr Ser
Ala His Met His Cys Val Trp Arg Ile Ser 20 25 30 Val Thr Pro Gly
Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp Leu 35 40 45 Tyr Arg
Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp Gly 50 55 60
Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys 65 70 75 47 76 PRT
Homo sapiens 47 Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln
Ser Pro Asn 1 5 10 15 Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys
Ile Trp Arg Ile Gln 20 25 30 Val Ser Glu Gly Phe His Val Gly Leu
Thr Phe Gln Ser Phe Glu Ile 35 40 45 Glu Arg His Asp Ser Cys Ala
Tyr Asp Tyr Leu Glu Val Arg Asp Gly 50 55 60 His Ser Glu Ser Ser
Thr Leu Ile Gly Arg Tyr Cys 65 70 75 48 76 PRT Homo sapiens 48 Cys
Gly Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly 1 5 10
15 Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val
20 25 30 Ala Pro Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe
Glu Thr 35 40 45 Glu Gly Asn Asp Val Cys Lys Tyr Asp Phe Val Glu
Val Arg Ser Gly 50 55 60 Leu Thr Ala Asp Ser Lys Leu His Gly Lys
Phe Cys 65 70 75 49 71 PRT Homo sapiens 49 Ser Ile Pro Ile Pro Gln
Lys Leu Phe Gly Glu Val Thr Ser Pro Leu 1 5 10 15 Phe Pro Lys Pro
Tyr Pro Asn Asn Phe Glu Thr Thr Thr Val Ile Thr 20 25 30 Val Pro
Thr Gly Tyr Arg Val Lys Leu Val Phe Gln Gln Phe Asp Leu 35 40 45
Glu Pro Ser Glu Gly Cys Phe Tyr Asp Tyr Val Lys Ile Ser Ala Asp 50
55 60 Lys Lys Leu Gly Arg Phe Cys 65 70 50 75 PRT Homo sapiens 50
Cys Ser Ser Glu Tyr Thr Glu Ala Ser Gly Tyr Ile Ser Ser Leu Glu 1 5
10 15 Tyr Pro Arg Ser Tyr Pro Pro Asp Leu Arg Cys Asn Tyr Ser Ile
Arg 20 25 30 Val Glu Arg Gly Leu Thr Leu His Leu Lys Phe Leu Glu
Pro Phe Asp 35 40 45 Ile Asp Asp His Gln Gln Val His Cys Pro Tyr
Asp Gln Leu Gln Ile 50 55 60 Tyr Ala Asn Gly Lys Asn Ile Gly Glu
Phe Cys 65 70 75 51 45 PRT Homo sapiens 51 Gly Thr Tyr Pro Pro Ser
Tyr Asn Leu Thr Phe His Ser Ser Gln Asn 1 5 10 15 Val Leu Leu Ile
Thr Leu Ile Thr Asn Thr Glu Arg Arg His Pro Gly 20 25 30 Phe Glu
Ala Thr Phe Phe Gln Leu Pro Arg Met Ser Ser 35 40 45 52 42 PRT Homo
sapiens 52 Gly Glu Arg Ser Gln Phe Val Val Thr Ser Asn Ser Asn Lys
Ile Thr 1 5 10 15 Val Arg Phe His Ser Asp Gln Ser Tyr Thr Asp Thr
Gly Phe Leu Ala 20 25 30 Glu Tyr Leu Ser Tyr Asp Ser Ser Asp Pro 35
40 53 36 PRT Homo sapiens 53 Thr Asn Pro Gly Thr Ile Arg Ile Phe
Ser Asn Gln Val Thr Ala Thr 1 5 10 15 Phe Leu Ile Glu Ser Asp Glu
Ser Asp Tyr Val Gly Phe Asn Ala Thr 20 25 30 Tyr Thr Ala Phe 35 54
37 PRT Homo sapiens 54 Gly Pro Gly Pro Val Lys Asp Val Phe Ser Thr
Thr Asn Arg Met Thr 1 5 10 15 Val Leu Leu Ile Thr Asn Asp Val Leu
Ala Arg Gly Gly Phe Lys Ala 20 25 30 Asn Phe Thr Thr Gly 35 55 37
PRT Homo sapiens 55 Gly Ser Lys Leu Pro Glu Pro Ile Val Ser Thr Asp
Ser Arg Leu Trp 1 5 10 15 Val Glu Phe Arg Ser Ser Ser Asn Trp Val
Gly Lys Gly Phe Phe Ala 20 25 30 Val Tyr Glu Ala Ile 35 56 36 PRT
Homo sapiens 56 Gly Tyr Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser
Arg Leu Trp 1 5 10 15 Leu Lys Phe Val Ser Asp Gly Ser Ile Asn Lys
Ala Gly Phe Ala Val 20 25 30 Asn Phe Phe Lys 35 57 37 PRT Homo
sapiens 57 Gly Ser Glu Lys Pro Glu Val Ile Thr Ser Gln Tyr Asn Asn
Met Arg 1 5 10 15 Val Glu Phe Lys Ser Asp Asn Thr Val Ser Lys Lys
Gly Phe Lys Ala 20 25 30 His Phe Phe Ser Glu 35 58 52 PRT Homo
sapiens 58 Gly Gln Leu Gly Ser Pro Leu Gly Asn Pro Pro Gly Lys Lys
Glu Phe 1 5 10 15 Met Ser Gln Gly Asn Lys Met Leu Leu Thr Phe His
Thr Asp Phe Ser 20 25 30 Asn Glu Glu Asn Gly Thr Ile Met Phe Tyr
Lys Gly Phe Leu Ala Tyr 35 40 45 Tyr Gln Ala Val 50 59 37 PRT Homo
sapiens 59 Gly Lys Gln Arg Pro Pro Asp Leu Asp Thr Ser Ser Asn Ala
Val Asp 1 5 10 15 Leu Leu Phe Phe Thr Asp Glu Ser Gly Asp Ser Arg
Gly Trp Lys Leu 20 25 30 Arg Tyr Thr Thr Glu 35 60 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 60
Ile Val Gly Gly 1 61 7 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 61 Trp Pro Trp Gln Val Ser
Leu 1 5 62 7 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 62 Cys Gly Gly Ser Leu Ile Ser 1 5 63 8
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 63 Trp Val Val Ser Ala Ala His Cys 1 5 64 7 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 64 Asp Asn Asp Ile Ala Leu Leu 1 5 65 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 65
Pro Ile Cys Leu Pro 1 5 66 4 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 66 Phe Pro Ala Gly 1 67 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 67 Val Thr Gly Trp Gly 1 5 68 12 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 68 Asp Ser Cys
Gln Gly Asp Ser Gly Gly Pro Leu Val 1 5
10 69 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 69 Gly Val Val Ser Trp Gly 1 5 70 4 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 70 Gly Cys Ala Gln 1 71 9 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 71 Asn Arg Pro
Gly Val Tyr Thr Arg Val 1 5 72 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 72 Asp Gly Ser
Asp Glu 1 5 73 4 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 73 Phe Ser Ser Pro 1 74 6 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 74
Asp Tyr Val Glu Ile Arg 1 5 75 4 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 75 Ser Ser Asn
Arg 1 76 4 PRT Artificial Sequence Description of Artificial
Sequence Synthetic activation peptide 76 Ile Ile Gly Gly 1 77 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
linking peptide 77 Gly Gly Gly Gly Ser 1 5 78 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic endoplasmic
retention sequence 78 Arg Glu Asp Leu Lys 1 5 79 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic endoplasmic
retention sequence 79 Arg Glu Asp Leu 1 80 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic endoplasmic
retention sequence 80 Arg Asp Glu Leu 1 81 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic endoplasmic
retention sequence 81 Lys Asp Glu Leu 1 82 10 PRT Artificial
Sequence Description of Artificial Sequence Synthetic His tag 82
Met Arg Gly Ser His His His His His His 1 5 10 83 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic activation
sequence 83 Val Val Gly Gly Thr 1 5 84 23 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer modified_base
(6) Deoxyinosine modified_base (9) Deoxyinosine modified_base (12)
Deoxyinosine modified_base (15) Deoxyinosine modified_base (18)
Deoxyinosine 84 tggrtnvtnw sngcnrcnca ytg 23 85 8 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide
MOD_RES (2) Ile or Val MOD_RES (3) Ile, Val, Leu or Met MOD_RES (4)
Ser or Thr MOD_RES (6) Asn or Thr 85 Trp Xaa Xaa Xaa Ala Xaa His
Cys 1 5 86 30 DNA Artificial Sequence Description of Artificial
Sequence Synthetic primer modified_base (1) Deoxyinosine
modified_base (4) Deoxyinosine modified_base (7) Deoxyinosine
modified_base (10) Deoxyinosine modified_base (16) Deoxyinosine
modified_base (21) Deoxyinosine modified_base (25) Deoxyinosine 86
nggnccnccn swrtcnccyt nrcanghrtc 30 87 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide MOD_RES (2)
Asn, Ser or Thr MOD_RES (4) Lys, Glu, Gln or His 87 Asp Xaa Cys Xaa
Gly Asp Ser Gly Gly Pro 1 5 10 88 7 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 88
accatgg 7 89 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 89 Asp Asp Asp Asp Lys 1 5 90 4 PRT Homo
sapiens 90 Val His Asp Ser 1 91 4 PRT Homo sapiens 91 Ile His Asp
Ser 1 92 110 PRT Homo sapiens 92 Cys Asp Gly Arg Phe Leu Leu Thr
Gly Ser Ser Gly Ser Phe Gln Ala 1 5 10 15 Thr His Tyr Pro Lys Pro
Ser Glu Thr Ser Val Val Cys Gln Trp Ile 20 25 30 Ile Arg Val Asn
Gln Gly Leu Ser Ile Lys Leu Ser Phe Asp Asp Phe 35 40 45 Asn Thr
Tyr Tyr Thr Asp Ile Leu Asp Ile Tyr Glu Gly Val Gly Ser 50 55 60
Ser Lys Ile Leu Arg Ala Ser Ile Trp Glu Thr Asn Pro Gly Thr Ile 65
70 75 80 Arg Ile Phe Ser Asn Gln Val Thr Ala Thr Phe Leu Ile Glu
Ser Asp 85 90 95 Glu Ser Asp Tyr Val Gly Phe Asn Ala Thr Tyr Thr
Ala Phe 100 105 110 93 111 PRT Homo sapiens 93 Cys Gly Gly Pro Phe
Glu Leu Trp Glu Pro Asn Thr Thr Phe Ser Ser 1 5 10 15 Thr Asn Phe
Pro Asn Ser Tyr Pro Asn Leu Ala Phe Cys Val Trp Ile 20 25 30 Leu
Asn Ala Gln Lys Gly Lys Asn Ile Gln Leu His Phe Gln Glu Phe 35 40
45 Asp Leu Glu Asn Ile Asn Asp Val Val Glu Ile Arg Asp Gly Glu Glu
50 55 60 Ala Asp Ser Leu Leu Leu Ala Val Tyr Thr Gly Pro Gly Pro
Val Lys 65 70 75 80 Asp Val Phe Ser Thr Thr Asn Arg Met Thr Val Leu
Leu Ile Thr Asn 85 90 95 Asp Val Leu Ala Arg Gly Gly Phe Lys Ala
Asn Phe Thr Thr Gly 100 105 110 94 113 PRT Homo sapiens 94 Cys Gly
Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro Glu 1 5 10 15
Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile Ser 20
25 30 Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp
Leu 35 40 45 Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val
Arg Asp Gly 50 55 60 Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe
Cys Gly Ser Lys Leu 65 70 75 80 Pro Glu Pro Ile Val Ser Thr Asp Ser
Arg Leu Trp Val Glu Phe Arg 85 90 95 Ser Ser Ser Asn Trp Val Gly
Lys Gly Phe Phe Ala Val Tyr Glu Ala 100 105 110 Ile 95 112 PRT Homo
sapiens 95 Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser
Pro Asn 1 5 10 15 Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile
Trp Arg Ile Gln 20 25 30 Val Ser Glu Gly Phe His Val Gly Leu Thr
Phe Gln Ser Phe Glu Ile 35 40 45 Glu Arg His Asp Ser Cys Ala Tyr
Asp Tyr Leu Glu Val Arg Asp Gly 50 55 60 His Ser Glu Ser Ser Thr
Leu Ile Gly Arg Tyr Cys Gly Tyr Glu Lys 65 70 75 80 Pro Asp Asp Ile
Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys Phe Val 85 90 95 Ser Asp
Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe Lys 100 105 110
96 113 PRT Homo sapiens 96 Cys Gly Gly Phe Leu Thr Lys Leu Asn Gly
Ser Ile Thr Ser Pro Gly 1 5 10 15 Trp Pro Lys Glu Tyr Pro Pro Asn
Lys Asn Cys Ile Trp Gln Leu Val 20 25 30 Ala Pro Thr Gln Tyr Arg
Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr 35 40 45 Glu Gly Asn Asp
Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly 50 55 60 Leu Thr
Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys 65 70 75 80
Pro Glu Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val Glu Phe Lys 85
90 95 Ser Asp Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe
Ser 100 105 110 Glu 97 123 PRT Homo sapiens 97 Ser Ile Pro Ile Pro
Gln Lys Leu Phe Gly Glu Val Thr Ser Pro Leu 1 5 10 15 Phe Pro Lys
Pro Tyr Pro Asn Asn Phe Glu Thr Thr Thr Val Ile Thr 20 25 30 Val
Pro Thr Gly Tyr Arg Val Lys Leu Val Phe Gln Gln Phe Asp Leu 35 40
45 Glu Pro Ser Glu Gly Cys Phe Tyr Asp Tyr Val Lys Ile Ser Ala Asp
50 55 60 Lys Lys Leu Gly Arg Phe Cys Gly Gln Leu Gly Ser Pro Leu
Gly Asn 65 70 75 80 Pro Pro Gly Lys Lys Glu Phe Met Ser Gln Gly Asn
Lys Met Leu Leu 85 90 95 Thr Phe His Thr Asp Phe Ser Asn Glu Glu
Asn Gly Thr Ile Met Phe 100 105 110 Tyr Lys Gly Phe Leu Ala Tyr Tyr
Gln Ala Val 115 120 98 112 PRT Homo sapiens 98 Cys Ser Ser Glu Tyr
Thr Glu Ala Ser Gly Tyr Ile Ser Ser Leu Glu 1 5 10 15 Tyr Pro Arg
Ser Tyr Pro Pro Asp Leu Arg Cys Asn Tyr Ser Ile Arg 20 25 30 Val
Glu Arg Gly Leu Thr Leu His Leu Lys Phe Leu Glu Pro Phe Asp 35 40
45 Ile Asp Asp His Gln Gln Val His Cys Pro Tyr Asp Gln Leu Gln Ile
50 55 60 Tyr Ala Asn Gly Lys Asn Ile Gly Glu Phe Cys Gly Lys Gln
Arg Pro 65 70 75 80 Pro Asp Leu Asp Thr Ser Ser Asn Ala Val Asp Leu
Leu Phe Phe Thr 85 90 95 Asp Glu Ser Gly Asp Ser Arg Gly Trp Lys
Leu Arg Tyr Thr Thr Glu 100 105 110
* * * * *
References