Note: Descriptions are shown in the official language in which they were submitted.
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BONE MARROW-DERIVED SERUM PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of bone marrow-
derived
serum proteins and to the use of these sequences in the diagnosis, treatment,
and prevention of
cancer, immune disorders, infections, and vascular disorders.
BACKGROUND OF THE INVENTION
Bone marrow is the site of blood cell formation, or hematopoiesis, from birth
throughout
adulthood. Blood cells are comprised of diverse cell types including red blood
cells (erythrocytes)
and white blood cells (leukocytes), all of which are derived from a common
progenitor stem cell.
During hematopoiesis, the stem cell is stimulated to proliferate and
differentiate by specific growth
factors called colony-stimulating factors. Blood cell maturation then proceeds
through various
stages, each stage characterized by further commitment of an immature blood
cell to a specific,
IS terminally differentiated state. In addition to hematopoietic cells, bone
marrow also contains
blood vessels, nerves, fatty tissue., and stromai cells. Stromal cells produce
a supporting
meshwork of collagen fibers and other extracellular matrix components which
are important for
promoting the growth and differentiation of hematopoietic cells. Deregulation
of hematopoiesis
can lead to neoplastic conditions such as leukemia or lymphoma, while
insufficient hematopoiesis
can lead to anemia or immunodeficiency.
A novel protein, MSE55 I,marrow stromal/endothelial cell protein, 55
kilodaltons), has
been identified from human stromal cells (Bahou, W.F. et al. ( 1992) J. Biol.
Chem. 267:13986-
13992). MSE55 is specifically expressed in stromal cells and in endothelial
cells which line blood
vessels. Furthermore, MSE55 is detected at relatively high levels in the
serum, suggesting that
stromal cells and/or endothelial cells secrete MSE55 into the circulation.
MSE55 cDNA contains
a long 5~ untranslated region of about 350 base pairs followed by a 1,173-base
pair open reading
frame that potentially encodes a 391-amino acid polypeptide. The observed
molecular weight of
55 kilodaltons exceeds the predicted molecular weight of 42 kilodaltons,
suggesting that MSE55
may undergo post-translational modifications such as glycosylation. Although
MSE55 is secreted
into the serum, the predicted amino acid sequence does not contain a signal
peptide. Lack of a
signal peptide, however, is also observed in other serum proteins such as
plasminogen activator
inhibitor 2 and ovalbumin. Other features of the predicted MSE55 sequence
include a serine- and
glycine-rich N-terminal region, an internal region of proline- and alanine-
rich tandem repeats, and
two putative metal-binding motifs. MSE55 is cross-reactive with antibodies
against another
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stromal cell protein, hemonectin, which plays an important role in white blood
cell adhesion and
maturation. Likewise, MSE55 ma.y also play a similar role in white blood cell
hematopoiesis.
B-lymphocytes are one of several types of differentiated white blood cells
which play a
critical role in the immune respon:>e to microbial infections. B-lymphocytes
enter the bloodstream
from the bone marrow and populate the spleen, lymph nodes, and other lymphoid
organs. In these
organs, B-cells encounter and react to foreign antigens by expressing and
secreting antibodies into
the circulation. Antibodies, or immunoglobulins, recognize and bind to
antigens on the surface of
blood-borne microbes. Antigen bonding triggers an immune response which leads
to the
destruction of the microbe.
The prototypical antibody is a tetramer consisting of two identical heavy
polypeptide
chains (H-chains) and two identical light polypeptide chains (L-chains)
interlinked by disulfide
bonds. This arrangement confers the characteristic Y-shape to antibody
molecules. Antibodies
are classified based on their H-chain composition. The five antibody classes,
IgA, IgD, IgE, IgG
and IgM, are defined by the a, 8, E:, y, and p H-chain types, respectively.
There are two types of
L-chains, K and ~,, either of which may associate as a pair with any H-chain
pair. IgG, the most
common class of antibody found in the circulation, is tetrameric as described
above, while the
other classes of antibodies are generally variants or multimers of this basic
structure.
H-chains and L-chains each contain an N-terminal variable region and a C-
terminal
constant region. The sequence of the constant region, which consists of about
110 amino acids in
L-chains and about 330 or 440 amino acids in H-chains, is nearly identical
among H- or L-chains
of a particular class. However, the sequence of the variable region, which
consists of about 110
amino acids, differs among H- or :L-chains of a particular class. Within each
H- and L-chain
variable region are three hypervariable regions of extensive sequence
diversity, each consisting of
about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain
hypervariable regions
come together to form the antigen binding site. (Reviewed in Alberts, B. et
al. (1994) Molecular
Biologv of the Cell, Garland Publishing, New York NY, pp. 1206-1213, 1216-
1217.)
Recombinant DNA technology has enabled the production of antibodies engineered
for
use as therapeutic and diagnostic agents. For example, rodent antibodies
directed against human
disease-associated proteins can be "humanized" by replacing their constant
regions with those
from human antibodies (Junghans, R.P. et al. (1990) Cancer Res. 50:1495-1502).
The variable
regions of these humanized antibodies recognize human disease-associated
proteins, while the
constant regions activate downstream effectors and prevent the antibodies
themselves from being
recognized as foreign in a human host. Humanized antibodies have proved to be
effective
therapeutic agents for the prevention of transplant rejection in primate model
systems and for their
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anti-proliferative activity in breast tumor cell lines (Brown, P.S. et 41.
(1991 ) Proc. Natl. Acad. Sci.
USA 88:2663-2667).
The discovery of new bone marrow-derived serum proteins and the
polynucleotides
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of cancer, immune disorders, infections,
and vascular
disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, bone marrow-
derived serum
proteins, referred to collectively as "BMDSP" and individually as "BMDSP-1"
and "BMDSP-2."
In one aspect, the invention provides a substantially purified polypeptide
comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-2, and
fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to at least one of the amino acid sequences selected from the
group consisting of SEQ
ID NO:1-2, and fragments thereof. The invention also provides an isolated and
purified
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-2, and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID >\f0:1-2, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucleotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-2,
and fragments
thereof. The invention also provides an isolated and purified polynucleotide
having a sequence
which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ 1D NO:1-2, and
fragments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample
containing nucleic acids, the method comprising the steps of (a) hybridizing
the complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one
aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:3-4,
and fragments
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thereof. The invention further provides an isolated and purified
poi,~nucleotide variant having at
least 90% polynucleotide sequenc;e identity to the polynucleotide sequence
selected from the
group consisting of SEQ ID 3-4, and fragments thereof. The invention also
provides an isolated
and purified polynucleotide having a sequence which is complementary to the
polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:3-4, and
fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO: l -2, and fragments thereof. In another aspect,
the expression
vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising
the steps of: (a) culturing the host cell containing an expression vector
containing at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
ID NO:1-2, and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected
from the group consisting of SEQ ID NO:1-2, and fragments thereof. The
invention also provides
a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of BMDSP, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-2, and fragments thereof, in conjunction with a
suitable
pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of BMDSP, the method comprising administering
to a subject in
need of such treatment an effective amount of an antagonist of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2, and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
Figures lA, 1B, and 1C show the amino acid sequence (SEQ ID NO:1 ) and nucleic
acid
sequence (SEQ ID N0:3) of BMDSP-1. The alignment was produced using MACDNASIS
PRO
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software (Hitachi Software Engineering, S. San Francisco CA).
Figures 2A, 2B, 2C, 2D, 2 E, and 2F show the amino acid sequence (SEQ ID N0:2)
and
nucleic acid sequence (SEQ ID NO:4) of BMDSP-2.
Figures 3A and 3B show the amino acid sequence alignment between BMDSP-2
(1859631; SEQ ID N0:2) and amino acids 1 through 218 of MSE55 (GI 338033; SEQ
ID NO:S),
produced using the multisequence; alignment program of LASERGENE software
(DNASTAR,
Madison WI).
Table 1 shows the tools, programs, and algorithms used to analyze BMDSP, along
with
applicable descriptions, reference,, and threshold parameters.
DES(~RIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any machines, materials, and methods similar or equivalent
to those described
herein can be used to practice or test the present invention, the preferred
machines, materials and
methods are now described. All publications mentioned herein are cited for the
purpose of
describing and disclosing the cell lines, protocols, reagents and vectors
which are reported in the
publications and which might be used in connection with the invention. Nothing
herein is to be
construed as an admission that the; invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFI1~TITIONS
"BMDSP" refers to the amino acid sequences of substantially purified BMDSP
obtained
from any species, particularly a mammalian species, including bovine, ovine,
porcine, murine,
equine, and preferably the human species, from any source, whether natural,
synthetic,
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semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to BMDSP, increases
or
prolongs the duration of the effect of BMDSP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
BMDSP.
An "allelic variant" is an .alternative form of the gene encoding BMDSP.
Allelic variants
may result from at least one mutation in the nucleic acid sequence and may
result in altered
mRNAs or in polypeptides whose structure or function may or may not be
altered. Any given
natural or recombinant gene may have none, one, or many allelic forms. Common
mutational
changes which give rise to allelic variants are generally ascribed to natural
deletions, additions, or
substitutions of nucleotides. Each of these types of changes may occur alone,
or in combination
with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding BMDSP include those sequences with
deletions, insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the
same as BMDSP or a polypeptide with at least one functional characteristic of
BMDSP. Included
within this definition are polymorphisms which may or may not be readily
detectable using a
particular oligonucleotide probe of the polynucleotide encoding BMDSP, and
improper or
unexpected hybridization to allelic variants, with a locus other than the
normal chromosomal locus
for the polynucleotide sequence encoding BMDSP. The encoded protein may also
be "altered,"
and may contain deletions, insertions, or substitutions of amino acid residues
which produce a
silent change and result in a functionally equivalent BMDSP. Deliberate amino
acid substitutions
may be made on the basis of similarity in polarity, charge, solubility,
hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues, as long as the
biological or
immunological activity of BMDSP is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, positively charged amino acids may
include lysine and
arginine, and amino acids with uncharged polar head groups having similar
hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine; asparagine
and glutamine; serine
and threonine; and phenylalanine and tyrosine.
The terms "amino acid" or "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. In this context, "fragments," "immunogenic fragments," or
"antigenic
fragments" refer to fragments of BMDSP which are preferably at least 5 to
about 15 amino acids
in length, most preferably at least 14 amino acids, and which retain some
biological activity or
immunological activity of BMDSP. Where "amino acid sequence" is recited to
refer to an amino
acid sequence of a naturally occurring protein molecule, "amino acid sequence"
and like terms are
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not meant to limit the amino acid sequence to the complete native amino acid
sequence associated
with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which, when bound to BMDSP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of BMDSP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
which decrease the effect of BMI)SP.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab')Z, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind BMDSP polypeptides can be prepared using intact polypeptides or
using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g.., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e.,
an epitope)
that makes contact with a particular antibody. When a protein or a fragment of
a protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (given regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
The term "biologically active," refers to a protein having structural,
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active"
refers to the capability of the natural, recombinant, or synthetic BMDSP, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
specific antibodies.
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The terms "complementary" or "complementarily" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-~A 5'." Complementarily between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarily exists between the single stranded molecules. The degree
of
complementarily between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
amplification reactions, which depend upon binding between nucleic acid
strands, and in the
design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising polynucleotide sequences encoding
BMDSP or
fragments of BMDSP may be employed as hybridization probes. The probes may be
stored in
freeze-dried form and may be associated with a stabilizing agent such as a
carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution containing
salts (e.g., NaCI),
detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution,
dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Nonvalk CT)
in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
more than one Incyte Clone using a computer program for fragment assembly,
such as the
GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been
both
extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the
detection of
the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding BMDSP, by
northern analysis is indicative of the presence of nucleic acids encoding
BMDSP in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding BMDSP.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
poiynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen. by an alkyl, acyl, or amino group. A
derivative polynucleotide
encodes a polypeptide which retains at least one biological or immunological
function of the
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natural molecule. A derivative polypeptide is one modified by glyc~sylation,
pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide
from which it was derived.
The term "similarity" refc;rs to a degree of complementarity. There may be
partial
similarity or complete similarity. The word "identity" may substitute for the
word "similarity." A
partially complementary sequence that at least partially inhibits an identical
sequence from
hybridizing to a target nucleic acid is referred to as "substantially
similar." The inhibition of
hybridization of the completely complementary sequence to the target sequence
may be examined
using a hybridization assay (Southern or northern blot, solution
hybridization, and the like) under
conditions of reduced stringency. A substantially similar sequence or
hybridization probe will
compete for and inhibit the binding of a completely similar (identical)
sequence to the target
sequence under conditions of reduced stringency. This is not to say that
conditions of reduced
stringency are such that non-specific binding is permitted, as reduced
stringency conditions
require that the binding of two sequences to one another be a specific (i.e.,
a selective) interaction.
The absence of non-specific binding may be tested by the use of a second
target sequence which
lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).
In the absence of non-specific binding, the substantially similar sequence or
probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGALIGN program
(DNASTAR)
which creates alignments between two or more sequences according to methods
selected by the
user, e.g., the clustai method. (See, e.g., Higgins, D.G. and P.M. Sharp
(1988) Gene 73:237-244.)
The clustal algorithm groups sequences into clusters by examining the
distances between all pairs.
The clusters are aligned pairwise :and then in groups. The percentage
similarity between two
amino acid sequences, e.g., sequence A and sequence B, is calculated by
dividing the length of
sequence A, minus the number of gap residues in sequence A, minus the number
of gap residues
in sequence B, into the sum of the. residue matches between sequence A and
sequence B, times
one hundred. Gaps of low or of no similarity between the two amino acid
sequences are not
included in determining percenta~;e similarity. Percent identity between
nucleic acid sequences
can also be counted or calculated by other methods known in the art, e.g., the
Jotun Hein method.
(See, e.g., Hein, J. ( 1990) Methods Enzymol. 183:626-645.) Identity between
sequences can also
be determined by other methods known in the art, e.g., by varying
hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
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contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of
the elements
required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a
solid support (e.g., paper, membranes, filters, chips, pins or glass slides,
or any other appropriate
substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, ete. These conditions can be
characterized by
expression of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which
may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" or ";array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of BMDSP. For example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of BMDSP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may be single-stranded or
double-stranded
and may represent the sense or the antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:3-4, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:3-4 is useful in hybridization and
amplification technologies
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and in analogous methods that distinguish SEQ ID N0:3-4 from rela.ed
polynucleotide sequences.
A fragment of SEQ ID N0:3-4 is ;at least about 15-20 nucleotides in length.
The precise length of
the fragment of SEQ ID N0:3-4 and the region of SEQ ID N0:3-4 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
S purpose for the fragment. In some; cases, a fragment, when translated, would
produce
polypeptides retaining some functional characteristic, e.g., antigenicity, or
structural domain
characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" or "operably linked" refer to functionally
related nucleic-
acid sequences. A promoter is operably associated or operably linked with a
coding sequence if
the promoter controls the translation of the encoded polypeptide. While
operably associated or
operably linked nucleic acid sequences can be contiguous and in the same
reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the
polypeptide but still bind to operator sequences that control expression of
the polypeptide.
The term "oligonucleotide:" refers to a nucleic acid sequence of at least
about 6 nucleotides
to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably
about 20 to 25
nucleotides, which can be used in PCR amplification or in a hybridization
assay or microarray.
"Oligonucleotide" is substantially equivalent to the terms "amplimer,"
"primer," "oligomer," and
"probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at: least about S nucleotides in length linked
to a peptide backbone
of amino acid residues ending in lysine. The terminal lysine confers
solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding BMDSP, or fragments thereof, or BMDSP itself, may comprise a
bodily fluid; an
extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic
DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue
print; etc.
The terms "specific binding" or "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, .an antibody, or an antagonist. The
interaction is dependent upon
the presence of a particular structure of the protein, e.g., the antigenic
determinant or epitope,
recognized by the binding molecule. For example, if an antibody is specific
for epitope "A," the
presence of a polypeptide containing the epitope A, or the presence of free
unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the amount of
labeled A that binds
to the antibody.
CA 02345881 2001-03-30
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The term "stringent condiaions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of formamide, or raising
the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60%-
free, preferably about 75% free, and most preferably about 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to
various methods well known in the art, and may rely on any known method for
the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The
method for
transformation is selected based on the type of host cell being transformed
and may include, but is
not limited to, viral infection, elec;troporation, heat shock, lipofection,
and particle bombardment.
The term "transformed" cells includes stably transformed cells in which the
inserted DNA is
capable of replication either as an autonomously replicating plasmid or as
part of the host
chromosome, as well as transiently transformed cells which express the
inserted DNA or RNA for
limited periods of time.
A "variant" of BMDSP polypeptides refers to an amino acid sequence that is
altered by
one or more amino acid residues. The variant may have "conservative" changes,
wherein a
substituted amino acid has similar structural or chemical properties (e.g.,
replacement of leucine
with isoleucine). More rarely, a variant may have "nonconservative" changes
(e.g., replacement of
glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
computer programs well known in the art, for example, LASERGENE software
(DNASTAR).
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WO 00/20588 PCT/US99/22908
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a polynucleotide sequence related to BMDSP. This definition may also
include, for
example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternate splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or an
absence of domains.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have <.~ignificant amino acid identity relative to
each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
THE INVENTION
The invention is based on the discovery of new human bone marrow-derived serum
proteins (BMDSP), the polynucleotides encoding BMDSP, and the use of these
compositions for
the diagnosis, treatment, or prevention of cancer, immune disorders,
infections, and vascular
disorders.
Nucleic acids encoding the BMDSP-1 ofthc present invention were identified in
lncyte
Clone 135698H1 from the bone marrow cDNA library (BMARNOT02) using a computer
search
for nucleotide and/or amino acid sequence alignments. A consensus sequence,
SEQ ID N0:3, was
derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones
135698H1 (BMARNOT02), 1320039H1 (BLADNOT04), 79242471 (PROSTUT03), 343067576
(SKINNOT04), and 2056224X 14R 1 (BEPINOTO 1 ).
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid
sequence of SEQ ID NO:1, as shown in Figures lA, 1B, and 1C. BMDSP-1 is 234
amino acids in
length and has eight potential casein kinase II phosphorylation sites at 718,
534, S87, 596, 7122,
5182, 7184, and S202 and two potential protein kinase C phosphorylation sites
at S42 and 572.
PFAM analysis indicates that BMDSP-1 contains two immunoglobulin domains from
G36 to
Q110 and from S 147 to V216. W ithin and overlapping the latter domain are
four immunoglobulin
signatures as indicated by BLOCIS;S, MOTIFS, and PROFILESCAN analyses. These
signatures
include amino acid residues from D190 to E233, from S151 to A173, and from
Y212 to F229.
Likewise, BLAST searches of protein databases indicate that BMDSP-1 has
chemical and
structural similarity with immunol;lobulin x light chain. A fragment of SEQ ID
N0:3 from about
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WO 00/20588 PCT/US99/22908
nucleotide 339 to about nucleotide 407 is useful in hybridization or
amplification technologies to
identify SEQ ID N0:3 and to distinguish between SEQ ID N0:3 and a related
sequence. Northern
analysis shows the expression of this sequence in various libraries, at least
63% of which are
associated with cancer or cell proliferation and at least 37% of which are
associated with
inflammation or trauma. In particular, 26% of the libraries expressing BMDSP-1
are derived from
gastrointestinal tissue, and 26% are derived from reproductive tissue.
Nucleic acids encoding the BMDSP-2 of the present invention were identified in
Incyte
Clone 1859631H1 from the prostate cDNA library (PROSNOTI 8) using a computer
search for
nucleotide and/or amino acid sequence alignments. A consensus sequence, SEQ ID
N0:4, was
derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones
1859631H1 (PROSNOT18), 1426610F1 (SINTBSTO1), 1511409F1 (LUNGNOT14), 1349506F1
(LATRTUT02), 1005112H1 (BRSTNOT03), 198094176 (LUNGTUT03), 121378581
(BRSTTUTOI ), 154443381 (PROSTUT04), and 1440896F 1 (THYRNOT03).
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid
I S sequence of SEQ ID N0:2, as shown in Figures 2A, 2B, 2C, 2D, 2E, and 2F.
BMDSP-2 is 254
amino acids in length and has two potential N-glycosylation sites at N88 and N
162 and eight
potential casein kinase II phosphorylation sites at S89, S 130, S 158, S 191,
7209, 7218, S223, and
5241. As shown in Figures 3A and 3B, BMDSP-2 has chemical and structural
similarity with
MSE55. BMDSP-2 shares 25% amino acid sequence identity with the N-terminal
region of
MSESS from amino acids I through 218 (GI 338033; SEQ ID NO:S). In particular,
the region of
BMDSP-2 from L26 to G68 shares 60% identity with the homologous region of
MSE55 and
contains six glycine residues, consistent with the glycine-rich nature of the
MSE55 N-terminal
region. Furthermore, nine out of eleven residues which comprise the second
putative metal
binding motif in MSE55 are conserved in BMDSP-2 from Q235 to E245.
Like the eDNA encoding MSE55, the cDNA encoding BMDSP-2 (SEQ ID N0:4)
contains a long 5' untranslated region of about 415 base pairs. A fragment of
SEQ ID N0:4 from
about nucleotide 383 to about nu~:leotide 442 is useful in hybridization or
amplification
technologies to identify SEQ ID N0:4 and to distinguish between SEQ ID N0:4
and a related
sequence. Northern analysis shows the expression of this sequence in various
libraries, at least
67% of which are associated with cancer or cell proliferation and at least 34%
of which are
associated with inflammation or trauma. In particular, 27% of the libraries
expressing BMDSP-2
are derived from reproductive tissue, and 20% are derived from cardiovascular
tissue.
The invention also encompasses BMDSP variants. A preferred BMDSP variant is
one
which has at least about 80%, more preferably at least about 90%, and most
preferably at least
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WO 00/20588 PCT/US99/22908
about 95% amino acid sequence identity to the BMDSP amino acid szquence, and
which contains
at least one functional or structural characteristic of BMDSP.
The invention also encompasses polynucleotides which encode BMDSP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:3-4, which encodes BMDSP.
The invention also encompasses a variant of a polynucleotide sequence encoding
BMDSP.
In particular, such a variant polynucleotide sequence will have at least about
80%, more preferably
at least about 90%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding BMDSP. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:3-4 which has at least about 80%, more preferably at least about
90%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:3-4. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of BMDSP.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynncleotide sequences encoding BMDSP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring BMDSP, and all such variations
are to be
considered as being specifically disclosed.
Although nucleotide sequences which encode BMDSP and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
BMDSP under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding BMDSP or its derivatives possessing a substantially
different codon usage,
e.g., inclusion of non-naturally occurring codons. Codons may be selected to
increase the rate at
which expression of the peptide occurs in a particular prokaryotic or
eukaryotic host in accordance
with the frequency with which particular codons are utilized by the host.
Other reasons for
substantially altering the nucleotide sequence encoding BMDSP and its
derivatives without
altering the encoded amino acid sequences include the production of RNA
transcripts having more
desirable properties, such as a greater half life, than transcripts produced
from the naturally
occurring sequence.
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
The invention also encompasses production of DNA sequences which encode BMDSP
and BMDSP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production,
the synthetic sequence may be inserted into any ofthe many available
expression vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding BMDSP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:3 and SEQ ID N0:4 and fragments thereof under various conditions of
stringency. (See, e.g.;
Wahl, G.M. and S.L. Berger (198'7) Methods Enzymol. 152:399-407; Kimmel, A.R.
(1987)
Methods Enzymol. 152:507-511.) For example, stringent salt concentration will
ordinarily be less
than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about
500 mM NaCI
and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI
and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in the absence
of organic solvent,
e.g., formamide, while high stringency hybridization can be obtained in the
presence of at least
about 35% formamide, and most preferably at least about 50% formamide.
Stringent temperature
conditions wilt ordinarily include temperatures of at least about 30°C,
more preferably of at least
about 37°C, and most preferably of at least about 42°C. Varying
additional parameters, such as
hybridization time, the concentration of detergent, e.g., sodium dodecyl
sulfate (SDS), and the
inclusion or exclusion of carrier IrINA, are well known to those skilled in
the art. Various levels of
stringency are accomplished by combining these various conditions as needed.
In a preferred
embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM
trisodium citrate, and 1%
SDS. In a more preferred embodiment, hybridization will occur at 37°C
in 500 mM NaCI, 50 mM
trisodium citrate, 1% SDS, 35% formamide, and 100 ~g/m) denatured salmon sperm
DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at
42°C in 250 mM NaCI, 25
mM trisodium citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful
variations on
these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C:, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/Z2908
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate,
and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any
of the embodiments of the invention. The methods may employ such enzymes as
the Klenow
fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerase
(Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or:
combinations of polymerases and proofreading exonucleases such as those found
in the
ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence
preparation is automated with machines such as the MICROLAB 2200 (Hamilton,
Reno NV),
Peltier Thermal Cycler 200 (PTC'.200; MJ Research, Watertown MA) and the ABI
CATALYST
800 (Perkin-Elmer). Sequencing is then carried out using either ABI 373 or 377
DNA sequencing
systems (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are vvell known in the art. (See; e.g., Ausubel,
F.M. (1997) Short
Protocols in Molecular Biolo~y, .John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A.
(1995) Molecular Biology and Bioteehnoioey, Wiley VCH, New York NY, pp. 856-
853.)
The nucleic acid sequences encoding BMDSP may be extended utilizing a partial
nucleotide sequence and employing various PCR-based methods known in the art
to detect
upstream sequences, such as promoters and regulatory elements. For example,
one method which
may be employed, restriction-site; PCR, uses universal and nested primers to
amplify unknown
sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G.
(1993) PCR Methods
Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in
divergent
directions to amplify unknown sequence from a circularized template. The
template is derived
from restriction fragments comprising a known genomic locus and surrounding
sequences. (See,
e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method,
capture PCR, involves
PCR amplification of DNA fragments adjacent to known sequences in human and
yeast artificial
chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
1:1 I 1-119.) In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the
art. (See, e.g.,
Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one
may use PCR,
nested primers, and PROMOTEh',FINDER libraries (Clontech, Palo Alto CA) to
walk genomic
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
DNA. This procedure avoids the need to screen libraries and is use~ul in
finding intron/exon
junctions. For all PCR-based methods, primers may be designed using
commercially available
software, such as OLIGO 4.06 Primer Analysis software (National Biosciences,
Plymouth MN) or
another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of
S about 50% or more, and to anneal to the template at temperatures of about
68°C to 72°C.
When screening for full-llength cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) -
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular,
capillary sequencing may employ flowable polymers for electrophoretic
separation, four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection of the emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-
Elmer),
and the entire process from loading of samples to computer analysis and
electronic data display
may be computer controlled. Capillary electrophoresis is especially preferable
for sequencing
small DNA fragments which may be present in limited amounts in a particular
sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode BMDSP may be cloned in recombinant DNA molecules that direct
expression of
BMDSP, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the
inherent degeneracy of the genetic code, other DNA sequences which encode
substantially the
same or a functionally equivalent amino acid sequence may be produced and used
to express
BMDSP.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter BMDSP-encoding sequences for a
variety of purposes
including, but not limited to, modification of the cloning, processing, and/or
expression of the
gene product. DNA shuffling by random fragmentation and PCR reassembly of gene
fragments
and synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example,
oligonucleotide-mediated site-directed mutagenesis may be used to introduce
mutations that create
new restriction sites, alter glycosyiation patterns, change codon preference,
produce splice
variants, and so forth.
In another embodiment, sequences encoding BMDSP may be synthesized, in whole
or in
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
part, using chemical methods well known in the art. (See, e.g., Can.thers,
M.H, et al. (1980)
Nucleic Acids Symp. Ser. 7:215-:223; Horn, T. et al. (1980) Nucleic Acids
Symp. Ser. 7:225-232.)
Alternatively, BMDSP itself or a fragment thereof may be synthesized using
chemical methods.
For example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using
the ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of
BMDSP, or any part thereof, may be altered during direct synthesis and/or
combined with
sequences from other proteins, or any part thereof, to produce a variant
polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-
421.) The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active BMDSP, the nucleotide sequences
encoding
BMDSP or derivatives thereof may be inserted into an appropriate expression
vector, i.e., a vector
which contains the necessary elements for transcriptional and translational
control of the inserted
coding sequence in a suitable host. These elements include regulatory
sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3' untranslated
regions in the vector
and in polynucleotide sequences encoding BMDSP. Such elements may vary in
their strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding BMDSP. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding BMDSP
and its initiation
codon and upstream regulatory sequences are inserted into the appropriate
expression vector, no
additional transcriptional or translational control signals may be needed.
However, in cases where
only coding sequence, or a fragment thereof, is inserted, exogenous
translational control signals
including an in-frame ATG initiation codon should be provided by the vector.
Exogenous
translational elements and initiation codons may be of various origins, both
natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers
appropriate for the
particular host cell system used. (See, e.g., Scharf, D. et al. {1994) Results
Probl. Cell Differ.
20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct
expression vectors containing sequences encoding BMDSP and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo l;enetic recombination. (See, e.g.,
Sambrook, J. et al. (1989)
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
Molecular Clonin;~. A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo y,
John Wiley & Sons,
New York NY, ch. 9, 13, and 16. )
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding BMDSP. These include, but are not limited to,
microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression
vectors; yeast transformed with yeast expression vectors; insect cell systems
infected with viral
expression vectors (e.g., baculovirus); plant cell systems transformed with
viral expression vectors
(e.g., cauliflower mosaic virus, C~aMV, or tobacco mosaic virus, TMV) or with
bacterial
expression vectors (e.g., Ti or pB:R322 plasmids); or animal cell systems. The
invention is not
limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected
depending upon the use intended for polynucleotide sequences encoding BMDSP.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding BMDSP can
be achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla
CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding
BMDSP into the
vector's multiple cloning site disrupts the IacZ gene, allowing a colorimetric
screening procedure
for identification of transformed bacteria containing recombinant molecules.
In addition, these
vectors may be useful for in vitro transcription, dideoxy sequencing, single
strand rescue with
helper phage, and creation of nested deletions in the cloned sequence. (See,
e.g., Van Heeke, G.
and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities
of BMDSP are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of
BMDSP may be used. For example, vectors containing the strong, inducible TS or
T7
bacteriophage promoter may be used.
Yeast expression systems may be used for production of BMDSP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH,
may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors
direct either the secretion or intracellular retention of expressed proteins
and enable integration of
foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A, et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of BMDSP. Transcription of
sequences
encoding BMDSP may be driven viral promoters, e.g., the 355 and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987)
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
EMBO J. 6:307-31 I ). Alternatively, plant promoters such as the small subunit
of RUBISCO or
heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
Science and Technoloev (1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding BMDSP
may be ligated~
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a na~n-essential E1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses BMDSP in host cells. (See, e.g., Logan,
J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription
enhancers, such as
the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in
mammalian host
cells. SV40 or EBV-based vectors may also be used for high-level protein
expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat.
Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of BMDSP in cell lines is preferred. For example, sequences
encoding BMDSP can be
transformed into cell lines using e:rcpression vectors which may contain viral
origins of replication
and/or endogenous expression elements and a selectable marker gene on the same
or on a separate
vector. Following the introduction of the vector, cells may be allowed to grow
for about 1 to 2
days in enriched media before being switched to selective media. The purpose
of the selectable
marker is to confer resistance to a selective agent, and its presence allows
growth and recovery of
cells which successfully express the introduced sequences. Resistant clones of
stably transformed
cells may be propagated using tissue culture techniques appropriate to the
cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk or apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers
resistance to methotrexate; neo confers resistance to the aminoglycosides,
neomycin and G-418;
21
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
and als and pat confer resistance to chlorsulfuron and phosphinotriccn
acetyltransferase,
respectively. (See, e.g., Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA
77:3567-3570;
Colbere-Garapin, F. et al. (1981) :f. Mol. Biol. 150:1-14.) Additional
selectable genes have been
described, e.g., trpB and hisD, which alter cellular requirements for
metabolites. (See, e.g.,
Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-
8051.) Visible
markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), f3
glucuronidase and its
substrate f3-glucuronide, or luciferase and its substrate luciferin may be
used. These markers can
be used not only to identify transf~rmants, but also to quantify the amount of
transient or stable
protein expression attributable to a specific vector system. (See, e.g.,
Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of
interest is also present, the presence and expression of the gene may need to
be confirmed. For
example, if the sequence encoding; BMDSP is inserted within a marker gene
sequence,
transformed cells containing sequences encoding BMDSP can be identified by the
absence of
marker gene function. Alternatively, a marker gene can be placed in tandem
with a sequence
encoding BMDSP under the control of a single promoter. Expression of the
marker gene in
response to induction or selection usually indicates expression of the tandem
gene as well.
In general, host cells that contain the nucleic acid sequence encoding BMDSP
and that
express BMDSP may be identified by a variety of procedures known to those of
skill in the art.
These procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane,
solution, or chip based technologies for the detection and/or quantification
of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of BMDSP
using
either specific poIyclonal or monoclonal antibodies are known in the art.
Examples of such
techniques include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs),
and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay
utilizing monoclonal antibodies reactive to two non-interfering epitopes on
BMDSP is preferred,
but a competitive binding assay may be employed. These and other assays are
well known in the
art. (See, e.g., Hampton, R. et al. (1990) Serological Methods a Laboratory
Manual, APS Press,
St. Paul MN, Sect. IV; Coligan, J.I~. et al. (1997) Current Protocols in
Immunoloay, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998)
Immunochemical
Protocols, Humans Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art
22
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
and may be used in various nucleic acid and amino acid assays. Means for
producing labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding BMDSP
include oligolabeling, nick transl;ition, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding BMDSP, or any fragments
thereof, may be
cloned into a vector for the production of an mRNA probe. Such vectors are
known in the art, are
commercially available, and may be used to synthesize RNA probes in vitro by
addition of an
appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially available kits, such as those
provided by
Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable
reporter
molecules or labels which may bc; used for ease of detection include
radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors,
magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding BMDSP may be
cultured
under conditions suitable for the expression and recovery of the protein from
cell culture. The
protein produced by a transformed cell may be secreted or retained
intracellularly depending on
the sequence and/or the vector used. As will be understood by those of skill
in the art, expression
vectors containing polynucleotide;s which encode BMDSP may be designed to
contain signal
sequences which direct secretion of BMDSP through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression ofthe
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and a.cylation. Post-translational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are
available from
the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding BMDSP may be ligated to a heterologous sequence resulting
in translation of
a fusion protein in any of the aforementioned host systems. For example, a
chimeric BMDSP
protein containing a heterologous moiety that can be recognized by a
commercially available
antibody may facilitate the screening of peptide libraries for inhibitors of
BMDSP activity.
Heterologous protein and peptide moieties may also facilitate purification of
fusion proteins using
commercially available affinity matrices. Such moieties include, but are not
limited to, glutathione
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WO 00/20588 PCT/US99/22908
S-transferase (GST), maltose binding protein (MBP), thioredoxin (T, x),
calmodulin binding
peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP,
and 6-His
enable purification oftheir cognate fusion proteins on immobilized
glutathione, maltose,
phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG,
c-myc, and
hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using
commercially
available monoclonal and polyclonal antibodies that specifically recognize
these epitope tags. A
fusion protein may also be engineered to contain a proteolytic cleavage site
located between the
BMDSP encoding sequence and the heterologous protein sequence, so that BMDSP
may be
cleaved away from the heterologous moiety following purification. Methods for
fusion protein
expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of
commercially available kits may also be used to facilitate expression and
purification of fusion
proteins.
In a further embodiment of the invention, synthesis of radiolabeled BMDSP may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract systems
(Promega). These systems couple transcription and translation of protein-
coding sequences
operably associated with the T7, T'3, or SP6 promoters. Translation takes
place in the presence of
a radiolabeled amino acid precursor, preferably 'SS-methionine.
Fragments of BMDSP ma:y be produced not only by recombinant production, but
also by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
supra. pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-
Elmer). Various
fragments of BMDSP may be synthesized separately and then combined to produce
the full length
molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of BMDSP-1 and immunoglobulin x light chain. The expression of
BMDSP-1 is
closely associated with cancer and immune disorders. Therefore, BMDSP-1
appears to play a role
in cancer, immune disorders, and infections. Furthermore, chemical and
structural similarity
exists between regions of BMDSP-2 and MSE55. BMDSP-2 is expressed in
cardiovascular tissue
and tissues associated with cancer and immune disorders. Therefore, BMDSP-2
appears to play a
role in cancer, immune disorders, and vascular disorders. In the treatment of
cancer, immune
disorders, infections, and vascular disorders associated with increased BMDSP
expression or
activity, it is desirable to decrease the expression or activity of BMDSP. In
the treatment of
cancer, immune disorders, infections, and vascular disorders associated with
decreased BMDSP
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99122908
expression or activity, it is desirable to increase the expression or ac~ivity
of BMDSP.
Therefore, in one embodiment, BMDSP or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of BMDSP. Examples of such disorders include, but are not limited to,
cancers such as
adenocarcinoma, melanoma, sarcoma, teratocarcinoma, cancers of the adrenal
gland, bladder,
bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen,
testis, thymus, thyroid, and uterus, and in particular, hematopoietic cancers
such as lymphoma,
leukemia, and myeloma; immune disorders such as actinic keratosis, acquired
immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomeruionephritis, Goodpasture's
IS syndrome, gout, Graves' disease, JHashimoto's thyroiditis, paroxysmal
nocturnal hemoglobinuria,
hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia
with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia
gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis,
pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, and trauma;
infections such as those caused by viral agents classified as adenovirus,
arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,
orthomyxovirus,
parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus,
retrovirus, rhabdovirus,
and togavirus; infections caused b;y bacterial agents classified as
pneumococcus, staphylococcus,
streptococcus, bacillus, corynebacterium, clostridium, meningococcus,
gonococcus, listeria,
moraxella, kingella, haemophilus, legionella, bordetella, gram-negative
enterobacterium including
shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella,
francisella, yersinia,
bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia,
chlamydia, and
mycoplasma; infections caused by fungal agents classified as aspergillus,
blastomyces,
dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, and other
fungal agents
causing various mycoses; infections caused by parasites classified as
plasmodium or malaria-
causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma,
pneumocystis carinii,
CA 02345881 2001-03-30
WO 00/20588 PCTNS99/22908
intestinal protozoa such as giardia, trichomonas, tissue nematodes s,~ch as
trichinella, intestinal
nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as
schistosoma, and
cestrodes such as tapeworm; and vascular disorders such as arteriovenous
fistula, atherosclerosis
including atherosclerotic coronary artery disease, arteriosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis,
phlebothrombosis, vascular tumors, complications of thrombolysis, balloon
angioplasty, vascular
replacement, and coronary artery bypass, cardiovascular disease, heart
failure, heart disease,
angina pectoris, myocardial infarction, calcific aortic valve stenosis and
other aortic valve
disorders, endocarditis, carcinoid heart disease, and complications of cardiac
transplantation.
In another embodiment, a vector capable of expressing BMDSP or a fragment or
derivative thereof may be administered to a subject to treat or prevent a
disorder associated with
decreased expression or activity of BMDSP including, but not limited to, those
described above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified BMDSP in conjunction with a suitable pharmaceutical carrier may be
administered to a
subject to treat or prevent a disorder associated with decreased expression or
activity of BMDSP
including, but not limited to, those provided above.
In still another embodimc;nt, an agonist which modulates the activity of BMDSP
may be
administered to a subject to treat .or prevent a disorder associated with
decreased expression or
activity of BMDSP including, bul: not limited to, those listed above.
In a further embodiment, an antagonist of BMDSP may be administered to a
subject to
treat or prevent a disorder associated with increased expression or activity
of BMDSP. Examples
of such disorders include, but are not limited to, those cancers, immune
disorders, infections, and
vascular disorders described above. In one aspect, an antibody which
specifically binds BMDSP
may be used directly as an antagonist or indirectly as a targeting or delivery
mechanism for
bringing a pharmaceutical agent to cells or tissue which express BMDSP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding BMDSP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity o:f BMDSP including, but not limited to,
those described above.
In other embodiments, an,y of the proteins, antagonists, antibodies, agonists,
complementary sequences, or vectors of the invention may be administered in
combination with
other appropriate therapeutic agents. Selection of the appropriate agents for
use in combination
therapy may be made by one of ordinary skill in the art, according to
conventional pharmaceutical
principles. The combination of therapeutic agents may act synergistically to
effect the treatment
or prevention of the various disorders described above. Using this approach,
one may be able to
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
achieve therapeutic efficacy with lower dosages of each agent, thus .'educing
the potential for
adverse side effects.
An antagonist of BMDSF' may be produced using methods which are generally
known in
the art. In particular, purified BMDSP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind BMDSP.
Antibodies to BMDSP
may also be generated using methods that are well known in the art. Such
antibodies may include,
but are not limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab
fragments, and fragments produced by a Fab expression library. Neutralizing
antibodies (i.e.,
those which inhibit dimer formation) are especially preferred for therapeutic
use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice,
humans, and others may be immunized by injection with BMDSP or with any
fragment or
oligopeptide thereof which has irnmunogenic properties. Depending on the host
species, various
adjuvants may be used to increase; immunological response. Such adjuvants
include, but are not
limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum
are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
BMDSP have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides,
or fragments are identical to a portion of the amino acid sequence of the
natural protein and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
BMDSP amino acids may be fused with those of another protein, such as KLH, and
antibodies to
the chimeric molecule may be produced.
Monoclonal antibodies to BMDSP may be prepared using any technique which
provides
for the production of antibody molecules by continuous cell lines in culture.
These include, but
are not limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-
hybridoma technique. (See, e.g., ifCohler, G. et al. ( 1975) Nature 256:495-
497; Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci. USA
80:2026-2030; and Cole, S.P. et a:l. ( 1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984)
Proc. Natl. Acad. Sci. USA 81:68:11-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, technic,ues
described for the
production of single chain antibodies may be adapted, using methods known in
the art, to produce
BMDSP-specific single chain antibodies. Antibodies with related specificity,
but of distinct
idiotypic composition, may be generated by chain shuffling from random
combinatorial
immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad.
Sci. USA 88:10134-
10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents
as disclosed in the literature. (See;, e.g., Orlandi, R. et al. (1989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for BMDSP may also be
generated. For example, such fragments include, but are not limited to,
F(ab')2 fragments
produced by pepsin digestion of the antibody molecule and Fab fragments
generated by reducing
the disulfide bridges of the F(ab')2; fragments. Alternatively, Fab expression
libraries may be
constructed to allow rapid and easy identification of monoclonal Fab fragments
with the desired
specificity. (See, e.g., Huse, W.D.. et al. ( 1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with established
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
BMDSP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing
monoclonal antibodies reactive to two non-interfering BMDSP epitopes is
preferred, but a
competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for BMDSP.
Affinity is expressed as
an association constant, Ke, which is defined as the molar concentration of
BMDSP-antibody
complex divided by the molar concentrations of free antigen and free antibody
under equilibrium
conditions. The K, determined for a preparation of polyclonal antibodies,
which are
heterogeneous in their affinities for multiple BMDSP epitopes, represents the
average affinity, or
avidity, of the antibodies for BMDSP. The Ka determined for a preparation of
monoclonal
antibodies, which are monospecific; for a particular BMDSP epitope, represents
a true measure of
affinity. High-affinity antibody preparations with Ke ranging from about 109
to 10'2 L/mole are
preferred for use in immunoassays in which the BMDSP-antibody complex must
withstand
rigorous manipulations. Low-affinity antibody preparations with K, ranging
from about 106 to 10'
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
L/mole are preferred for use in immunopurification and similar provedures
which ultimately
require dissociation of BMDSP, preferably in active form, from the antibody
(Catty, D. ( 1988)
Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC; Liddell,
J.E. and Cryer,
A. ( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New
York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is preferred for use in procedures
requiring precipitation
of BMDSP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
and guidelines for antibody quality and usage in various applications, are
generally available.
(See, e.g., Catty, s_ upra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding BMDSP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding BMDSP may be used in situations in
which it would
be desirable to block the transcription of the mRNA. In particular, cells may
be transformed with
sequences complementary to polynucleotides encoding BMDSP. Thus, complementary
molecules
or fragments may be used to modulate BMDSP activity, or to achieve regulation
of gene function.
Such technology is now well known in the art, and sense or antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding BMDSP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses,
or from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the
targeted organ, tissue, or cell population. Methods which are well known to
those skilled in the art
can be used to construct vectors to express nucleic acid sequences
complementary to the
polynucleotides encoding BMDSP. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
Genes encoding BMDSP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding BMDSP.
Such constructs may be used to introduce untranslatable sense or antisense
sequences into a cell.
Even in the absence of integration into the DNA, such vectors may continue to
transcribe RNA
molecules until they are disabled 'by endogenous nucleases. Transient
expression may last for a
month or more with a non-replicating vector, and may last even longer if
appropriate replication
elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
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WO 00/20588 PCT/US99/22908
regulatory regions of the gene encoding BMDSP. Oligonucleotides ,derived from
the transcription
initiation site, e.g., between about: positions -10 and +10 from the start
site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes'. inhibition of the ability of the double
helix to open sufficiently
for the binding of polymerases, transcription factors, or regulatory
molecules. Recent therapeutic
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches,
Futura Publishing,
Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may
also be
designed to block translation of cr~RNA by preventing the transcript from
binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage
of RNA. The mechanism of ribozyme action involves sequence-specific
hybridization of the
ribozyme molecule to complementary target RNA, followed by endonucleolytic
cleavage. For
example, engineered hammerhead motif ribozyme molecules may specifically and
efficiently
catalyze endonucleolytic cleavage: of sequences encoding BMDSP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides, corresponding to the region of the target gene containing the
cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable. .
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of nucleic acid
molecules. These
include techniques for chemically synthesizing oligonucleotides such as solid
phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in vivo transcription of DNA sequences encoding BMDSP. Such DNA sequences
may be
incorporated into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or
SP6. Alternatively, these cDNA constructs that synthesize complementary RNA,
constitutively or
inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these; molecules by the inclusion of
nontraditional bases such as
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and
similarly modified forms
of adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by
endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally
suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors
may be introduced into
stem cells taken from the patient and clonally propagated for autologous
transplant back into that
same patient. Delivery by transfection, by liposome injections, or by
polycationic amino polymers
may be achieved using methods vrhich are well known in the art. (See, e.g.,
Goldman, C.K. et al.-
(1997)Nat. Biotechnol. 15:462-4~6.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of BMDSP, antibodies to BMDSF', and mimetics, agonists, antagonists, or
inhibitors of BMDSP.
The compositions may be administered alone or in combination with at least one
other agent, such
as a stabilizing compound, which may be administered in any sterile,
biocompatible
pharmaceutical carrier including, but not limited to, saline, buffered saline,
dextrose, and water.
The compositions may be administered to a patient alone, or in combination
with other agents,
drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intrarnuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Remin tg on';s Pharmaceutical Sciences (Maack
Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, for ingestion by
31
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WO 00/20588 PCT/US99/22908
the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or drag;ee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other
plants; cellulose, such as
methyl cellulose, hydroxypropylrnethyl-cellulose, or sodium
carboxymethylcelluiose; gums,
including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired,
disintegrating or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone,
agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated
sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft
capsules, the active compounds rnay be dissolved or suspended in suitable
liquids, such as fatty
oils, liquid, or liquid polyethylene; glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl
oieate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
manner that is known in the art, e.g., by means of conventional mixi.rg,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acid. Salts tend to be mare soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all of the following: 1 mM to 50 mM histidine,
0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to-
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
BMDSP, such labeling would include amount, frequency, and method of
administration.
Pharmaceutical compositions suitable for use in the invention include
compositions
wherein the active ingredients are contained in an effective amount to achieve
the intended
purpose. The determination of an effective dose is well within the capability
of those skilled in the
art.
For any compound, the therapeutically effective dose can be estimated
initially either in
cell culture assays, e.g., of neopla;stic cells or in animal models such as
mice, rats, rabbits, dogs, or
pigs. An animal model may also be used to determine the appropriate
concentration range and
rout~,of administration. Such information can then be used to determine useful
doses and routes
for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
BMDSP or fragments thereof, antibodies of BMDSP, and agonists, antagonists or
inhibitors of
BMDSP, which ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals,
such as by calculating the EDso (the dose therapeutically effective in SO% of
the population) or
LDso (the dose lethal to 50% of the population) statistics. The dose ratio of
toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LDS°/EDS° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies are used to formulate a range of dosage for
human use. The
dosage contained in such compositions is preferably within a range of
circulating concentrations
that includes the EDSO with little or no toxicity. The dosage varies within
this range depending
upon the dosage form employed, the sensitivity of the patient, and the route
of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
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CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
subject requiring treatment. Dosage and administration are adjususd to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account
include the severity of the disease state, the general health of the subject,
the age, weight, and
gender of the subject, time and frequency of administration, drug
combination(s), reaction
sensitivities, and response to therapy. Long-acting pharmaceutical
compositions may be
administered every 3 to 4 days, every week, or biweekly depending on the half
life and clearance
rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon t:he route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment.. antibodies which specifically bind BMDSP may be used
for the
diagnosis of disorders characterized by expression of BMDSP, or in assays to
monitor patients
being treated with BMDSP or agonists, antagonists, or inhibitors of BMDSP.
Antibodies useful
for diagnostic purposes may be prepared in the same manner as described above
for therapeutics.
Diagnostic assays for BMDSP include methods which utilize the antibody and a
label to detect
BMDSP in human body fluids or in extracts of cells or tissues. The antibodies
may be used with
or without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known
in the art and may be used.
A variety of protocols for measuring BMDSP, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of BMDSP
expression. Normal or standard values for BMDSP expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
BMDSP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
BMDSP expressed in subject, control, and disease samples from biopsied tissues
are compared
with the standard values. Deviation between standard and subject values
establishes the
parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding BMDSP may
be
used for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
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WO 00/20588 PCT/US99/22908
sequences, complementary RN,A and DNA molecules, and PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of BMDSP
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of BMDSP, and to monitor regulation of BMDSP
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding BMDSP or
closely related
molecules may be used to identify nucleic acid sequences which encode BMDSP.
The specificity
of the probe, whether it is made from a highly specific region, e.g., the S'
regulatory region, or
from a less specific region, e.g.., a conserved motif, and the stringency of
the hybridization or
amplification (maximal, high, intermediate, or low), will determine whether
the probe identifies
only naturally occurring sequences encoding BMDSP, allelic variants, or
related sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the BMDSP encoding sequences.
The hybridization
probes of the subject invention .may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:3-4 or from genomic sequences including promoters, enhancers, and
introns of the
BMDSP gene.
Means for producing specific hybridization probes for DNAs encoding BMDSP
include
the cloning of polynucleotide sequences encoding BMDSP or BMDSP derivatives
into vectors for
the production of mRNA probe s. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by means of the addition of
the appropriate
RNA polymerases and the appropriate labeled nucleotides. Hybridization probes
may be labeled
by a variety of reporter groups, for example, by radionuclides such as''-P or
35S, or by enzymatic
labels, such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and
the like.
Polynucleotide sequences encoding BMDSP may be used for the diagnosis of
disorders
associated with expression of B:MDSP. Examples of such disorders include, but
are not limited to,
cancers such as adenocarcinoma, melanoma, sarcoma, teratocarcinoma, cancers of
the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal
tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary
glands, skin, spleen, testis, thymus, thyroid, and uterus, and in particular,
hematopoietic cancers
such as lymphoma, leukemia, and myeloma; immune disorders such as actinic
keratosis, acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis,
CA 02345881 2001-03-30
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autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis,
cholecystitis,
cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes mellitus,
emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
paroxysmal nocturnal
hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome,
episodic lymphopenia
with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple
sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis,
pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, and trauma;
infections such as those caused by viral agents classified as adenovirus,
arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,
orthomyxovirus,
parvovirus, papovavirus, param:yxovirus, picornavirus, poxvirus, reovirus,
retrovirus, rhabdovirus,
and togavirus; infections caused by bacterial agents classified as
pneumococcus, staphylococcus,
streptococcus, bacillus, corynebacterium, clostridium, meningococcus,
gonococcus, listeria,
moraxella, kingella, haemophilus, legionella, bordetella, gram-negative
enterobacterium including
shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella,
francisella, yersinia,
bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia,
chlamydia, and
mycoplasma; infections caused by fungal agents classified as aspergillus,
blastomyces,
dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, and other
fungal agents
causing various mycoses; infections caused by parasites classified as
plasmodium or malaria-
causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma,
pneumocystis carinii,
intestinal protozoa such as giardia, trichomonas, tissue nematodes such as
trichinella, intestinal
nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as
schistosoma, and
cestrodes such as tapeworm; and vascular disorders such as arteriovenous
fistula, atherosclerosis
including atherosclerotic coronary artery disease, arteriosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis,
phlebothrombosis, vascular tumors, complications of thrombolysis, balloon
angioplasty, vascular
replacement, and coronary artery bypass, cardiovascular disease, heart
failure, heart disease,
angina pectoris, myocardial infarction, calcific aortic valve stenosis and
other aortic valve
disorders, endocarditis, carcinoid heart disease, and complications of cardiac
transplantation. The
polynucleotide sequences encoding BMDSP may be used in Southern or northern
analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in dipstick,
pin, and
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WO 00/20588 PCT/US99/22908
multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues
from patients to
detect altered BMDSP expression. Such qualitative or quantitative methods are
well known in the
art.
In a particular aspect, the nucleotide sequences encoding BMDSP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding BMDSP may be labeled by standard methods and
added to a fluid
or tissue sample from a patient under conditions suitable for the formation of
hybridization
complexes. After a suitable incubation period, the sample is washed and the
signal is quantitated
and compared with a standard value. If the amount of signal in the patient
sample is significantly
altered in comparison to a control sample then the presence of altered levels
of nucleotide
sequences encoding BMDSP in the sample indicates the presence of the
associated disorder. Such
assays may also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in
animal studies, in clinical trials, or to monitor the treatment of an
individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
BMDSP, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof; encoding BMDSP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially
purified polynucleotide is used. Standard values obtained in this manner may
be compared with
values obtained from samples from patients who are symptomatic for a disorder.
Deviation from
standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in
the patient begins to approximate that which is observed in the normal
subject. The results
obtained from successive assays may be used to show the efficacy of treatment
over a period
ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or rnay provide a means for detecting the disease
prior to the
appearance of actual clinical symptoms. A more definitive diagnosis of this
type may allow health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing
the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
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WO 00/20588 PCT/I3S99/22908
BMDSP may involve the use of PCR. These oligomers may be cuemically
synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a
polynucleotide encoding BMDSP, or a fragment of a polynucleotide complementary
to the
polynucieotide encoding BMDSP, and will be employed under optimized conditions
for
identification of a specific gene or condition. Oligomers may also be employed
under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of BMDSP include
radiolabeling or biotinylating nucleotides, coamplification of a control
nucleic acid, and
interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J. Immunol.
Methods 159:235-244; Duplaa., C. et al. (1993) Anal. Biochem. 212:229-236.)
The speed of
quantitation of multiple sample, may be accelerated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectrophotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
IS polynucleotide sequences described herein may be used as targets in a
microarray. The
microarray can be used to monitor the expression level of large numbers of
genes simultaneously
and to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, and
to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See,
e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl.
Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/2511 16;
Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997)
Proc. Natl. Acad.
Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding BMDSP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic
sequence. The sequences may be mapped to a particular chromosome, to a
specific region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial
P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997)
Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask,
B.J. (1991) Trends
Genet. 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich,
et al. (1995) in
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WO 00/20588 PCT/US99/22908
Meyers, supra, pp. 965-968.) >='sxamples of genetic map data can b,; found in
various scientific
journals or at the Online Mende,lian Inheritance in Man (OMIM) site.
Correlation between the
location of the gene encoding BMDSP on a physical chromosomal map and a
specific disorder, or
a predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such
as linkage analysis using established chromosomal markers, may be used for
extending genetic-
maps. Often the placement of a gene on the chromosome of another mammalian
species, such as
mouse, may reveal associated markers even if the number or arm of a particular
human
chromosome is not known. New sequences can be assigned to chromosomal arms by
physical
mapping. This provides valuable information to investigators searching for
disease genes using
positional cloning or other gene; discovery techniques. Once the disease or
syndrome has been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
IS l 1q22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-
580.) The nucleotide
sequence ofthe subject invention may also be used to detect differences in the
chromosomal
location due to translocation, inversion, etc., among normal, carrier, or
affected individuals.
In another embodiment of the invention, BMDSP, its catalytic or immunogenic
fragments,
or oligopeptides thereof can be used for screening libraries of compounds in
any of a variety of
drug screening techniques. The fragment employed in such screening may be free
in solution,
affixed to a solid support, borne. on a cell surface, or located
intracellularly. The formation of
binding complexes between BN(DSP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds having suitable binding affinity to the protein of interest. (See,
e.g., Geysen, et al.
( 1984) PCT application W084/03564.) In this method, large numbers of
different small test
compounds are synthesized on a solid substrate. The test compounds are reacted
with BMDSP, or
fragments thereof, and washed. Bound BMDSP is then detected by methods well
known in the
art. Purified BMDSP can also be coated directly onto plates for use in the
aforementioned drug
screening techniques. Alternativeiy, non-neutralizing antibodies can be used
to capture the
peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing antibodies capable of binding BMDSP specifically compete with a
test compound for
binding BMDSP. In this manner, antibodies can be used to detect the presence
of any peptide
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WO 00/20588 PCT/US99/22908
which shares one or more antigenic determinants with BMDSP.
In additional embodinrents, the nucleotide sequences which encode BMDSP may be
used
in any molecular biology techniques that have yet to be developed, provided
the new techniques
rely on properties of nucleotide sequences that are currently known,
including, but not limited to,
such properties as the triplet genetic code and specific base pair
interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of
the remainder of the disclosure; in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. [Attorney Docket No. PF-0609 P, filed October 2,
1998], are hereby
expressly incorporated by reference.
EXAMPLES
Construction of cDNA Libraries
BMARNOT02
The BMARNOT02 library was constructed using RNA purchased from Clontech. The
RNA was isolated from the borne marrow of 24 male and female Caucasian donors,
16 to 70 years
old. The cDNA library was custom constructed by Stratagene using this RNA.
cDNA synthesis
was primed using oligo d(T) and random hexamers, and the cDNA library was
cloned using the
UNIZAP vector system (Stratagene).
PROSNOTI 8
The PROSNOT18 library was constructed using RNA isolated from diseased
prostate
tissue removed from a 58-year-old Caucasian male during a radical cystectomy,
radical
prostatectomy, and gastrostomy. Pathology indicated adenofibromatous
hyperplasia. Pathology
for the associated tumor tissue indicated grade 3 transitional cell carcinoma.
Patient history
included angina and emphyserrra. Family history included acute myocardial
infarction,
atherosclerotic coronary artery disease, and type II diabetes.
Frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution
using a
Polytron PT-3000 homogenizes (Brinkmann Instruments, Westbury NY). The iysate
was
centrifuged over a CsCI cushion to isolate RNA. The RNA was extracted with
acid phenol,
precipitated with sodium acetate and ethanol, resuspended in RNase-free water,
and treated with
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
DNase. The RNA was re-extras;ted with acid phenol and reprecipi~ated as
described above.
Poly(A+) RNA was isolated using the OLIGOTEX mRNA purification kit (QIAGEN,
Chatsworth
CA).
Poly(A+) RNA was used for cDNA synthesis and construction of the cDNA library
according to the recommended protocols in the SUPERSCRIPT plasmid system (Life
Technologies). The cDNAs were fractionated on a SEPHAROSE CL4B column
(Amersham
Pharmacia Biotech), and those cDNAs exceeding 400 by were ligated into pINCY
(Incyte
Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into
DHSa competent
cells (Life Technologies).
II. Isolation of cDNA Clones
BMARNOT02
cDNA clones were recovered by in vivo excision as single-stranded PBLUESCRIPT
phagemids (Stratagene). These phagemids were used to reinfect SOLR host cells
(Stratagene)
from which double-stranded recombinant phagemids were purified using either
the QIAWELL-8
plasmid purification system (QhAGEN) or the MINIPREP plasmid purification kit
(Advanced
Genetic Technologies Corp., Gaithersburg, MD).
PROSNOT 18
Plasmid DNA was released from the cells and purified using the R.E.A.L. Prep
96 plasmid
kit (QIAGEN). The recommended protocol was employed except for the following
changes: I)
the bacteria were cultured in 1 rnl of sterile Terrific Broth (Life
Technologies) with carbenicillin at
mg/I and glycerol at 0.4%; 2) after the cultures were incubated for 19 hours,
the cells were
lysed with 0.3 ml of lysis buffer.; and 3} following isopropanol
precipitation, the plasmid DNA
pellets were each resuspended in 0.1 ml of distilled water. The DNA samples
were stored at 4°C.
III. Sequencing and Analysis
25 The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer)
or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton)
systems in
combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were
sequenced using
the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base
calling software, and kits. In one alternative, cDNAs were sequenced using the
MEGABACE
1000 DNA sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were
amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing
ready
reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced
using solutions
and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were
determined
using standard methods (reviewed in Ausubel, 1997, sera, unit 7.7). Some of
the cDNA
41
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
sequences were selected for exte;nsion using the techniques disclos;,d in
Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
sequencing
were assembled and analyzed using a combination of software programs which
utilize algorithms
well known to those skilled in the art. Table 1 summarizes the tools,
programs, and algorithms
used and provides applicable descriptions, references, and threshold
parameters. The first column
of Table I shows the tools, programs, and algorithms used, the second column
provides brief
descriptions, the third column presents appropriate references, all of which
are incorporated by
reference herein in their entirety, and the fourth column presents, where
applicable, the scores,
probability values, and other parameters used to evaluate the strength of a
match between two
sequences (the higher the score, the greater the homology between two
sequences). Sequences
were analyzed using MACDNASIS PRO software (Hitachi Software Engineering) and
LASERGENE software (DNAS'fAR).
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then
queried against a selection of public databases, such as the GenBank primate,
rodent, mammalian,
vertebrate, and eukaryote databases, and BLOCKS to acquire annotation using
programs based on
BLAST, FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide
sequences using programs based on Phred, Phrap, and Consed, and were screened
for open
reading frames using programs based on GeneMark, BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length amino acid
sequences, and these full length sequences were subsequently analyzed by
querying against
databases such as the GenBank databases (described above), SwissProt, BLOCKS,
PRINTS,
PFAM, and Prosite.
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from
SEQ ID N0:3-4. Fragments from about 20 to about 4000 nucleotides which are
useful in
hybridization and amplification technologies were described in The Invention
section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which
RNAs from a particular cell type or tissue have been bound. (See, e.g.,
Sambrook, supra, ch. 7;
Ausubel, 1995, supra, ch. 4 and 1:6.)
Analogous computer techniques applying BLAST were used to search for identical
or
42
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
related molecules in nucleotide databases such as GenBank or L,II-ESEQ (Incyte
Pharmaceuticals).
This analysis is much faster than multiple membrane-based hybridizations. In
addition, the
sensitivity of the computer search can be modified to determine whether any
particular match is
categorized as exact or similar. The basis of the search is the product score,
which is defined as:
sectuence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1% to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between 15 and 40,
although lower scores may identify related molecules.
The results of northern .analyses are reported as a percentage distribution of
libraries in
which the transcript encoding BMDSP occurred. Analysis involved the
categorization of cDNA
libraries by organ/tissue and disease. The organ/tissue categories included
cardiovascular,
IS dermatologic, developmental, endocrine, gastrointestinal,
hematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition categories included
cancer,
inflammation/trauma, cell proliferation, neurological, and pooled. For each
category, the number
of libraries expressing the sequence of interest was counted and divided by
the total number of
libraries across all categories. Percentage values of tissue-specific and
disease- or condition-
specific expression are reported in The Invention section.
V. Extension of BMDSP )H:ncoding Polynucleotides
The full length nucleic acid sequences of SEQ 1D N0:3-4 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from
this fragment. One primer was synthesized to initiate 5' extension of the
known fragment, and the
other primer, to initiate 3' extension of the known fragment. The initial
primers were designed
using OLIGO 4.06 software (National Biosciences), or another appropriate
program, to be about
22 to 30 nucleotides in length, to have a GC content of about 50% or more, and
to anneal to the
target sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human eDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz',
43
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
(NH,)ZS04, and (3-mercaptoethanol, Taq DNA polymerise (Ariremham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3
min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step S: Steps 2, 3,
and 4 repeated 20 times; Step 6:
S 68°C, S min; Step 7: storage at 4°C. In the alternative, the
parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 1S sec; Step
3: S7°C, 1 min; Step 4: 68°C, 2
min; Step S: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, S min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,
Eugene OR)
dissolved in 1X TE and O.S pl of undiluted PCR product into each well of an
opaque fluorimeter
plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The
plate was
scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the
fluorescence of the
sample and to quantify the concentration of DNA. A S ~l to 10 ~1 aliquot of
the reaction mixture
was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which
reactions were
successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJl cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Phar~macia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to
fill-in
restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were
selected on antibiotic-containing media, individual colonies were picked and
cultured overnight at
2S 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech.) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 1S sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step S: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low
DNA recoveries were reamplified using the same conditions as described above.
Samples were
diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer
sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or
the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
44
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
In like manner, the nucleotide sequences of SEQ ID N0.3-4 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:3-4 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20
base pairs, is specifically described, essentially the same procedure is used
with larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 /cCi of
['y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-
based hybridization analysis of human genomic DNA digested with one of the
following
endonucleases: Ase l, Bgl II, E<;o RI, Pst I, Xba I, or Pvu I I (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuelt, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. Hybridization patterns are visualized and compared using
autoradiography.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an elc;ment on the microarray may be assessed
through analysis of the
scanned images.
Fuli-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the elements of the microarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGENE software (DNASTAR).
Full-length
cDNAs, ESTs, or fragments then eof corresponding to one of the nucleotide
sequences of the
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
present invention, or selected a.t random from a cDNA library rel.,vant to the
present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed
to the slide using, e.g.,
UV cross-linking followed by thermal and chemical treatments and subsequent
drying. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome
Res. 6:639-645.)
Fluorescent probes are prepared and used for hybridization to the elements on
the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the BMDSP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring BMDSP.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides
are designed using OLIGO 4.Ofi software (National Biosciences) and the coding
sequence of
BMDSP. To inhibit transcription, a complementary oligonucleotide is designed
from the most
unique 5' sequence and used to prevent promoter binding to the coding
sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent ribosomal
binding to the
BMDSP-encoding transcript.
IX. Expression of BMDSP
Expression and purification of BMDSP is achieved using bacterial or virus-
based
expression systems. For expression of BMDSP in bacteria, cDNA is subcloned
into an
appropriate vector containing an antibiotic resistance gene and an inducible
promoter that directs
high levels of cDNA transcription. Examples of such promoters include, but are
not limited to, the
trp-lac (tac) hybrid promoter and the TS or T7 bacteriophage promoter in
conjunction with the lac
operator regulatory element. Recombinant vectors are transformed into suitable
bacterial hosts,
e.g., BL21(DE3). Antibiotic resistant bacteria express BMDSP upon induction
with isopropyl
beta-D-thiogalactopyranoside (IPTG). Expression of BMDSP in eukaryotic cells
is achieved by
infecting insect, or mammalian cell lines with recombinant Autographica
califomica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential
polyhedrin
gene of baculovirus is replaced with cDNA encoding BMDSP by either homologous
recombination or bacterial-mediated transposition involving transfer plasmid
intermediates. Viral
infectivity is maintained and the. strong polyhedrin promoter drives high
levels of cDNA
transcription. Recombinant baculovirus is used to infect Spodoptera fruQinerda
(S~) insect cells
in most cases, or human hepatoc;ytes, in some cases. Infection of the latter
requires additional
genetic modifications to baculovirus. (See Engelhard, E.K. et al. ( 1994)
Proc. Natl. Acad. Sci.
USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
46
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
In most expression systems, BMDSP is synthesized as a :usion protein with,
e.g.,
glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-
His, permitting rapid,
single-step, affinity-based purification of recombinant fusion protein from
crude cell lysates.
GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the
purification of fusion
proteins on immobilized glutathione under conditions that maintain protein
activity and
antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be
proteolytically cleaved from BMDSP at specifically engineered sites. FLAG, an
8-amino acid
peptide, enables immunoaffinity purification using commercially available
monoclonal and
polyclonal anti-FLAG antibodi'a (Eastman Kodak). 6-His, a stretch of six
consecutive histidine
residues, enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression
and purification are discussed in Ausubel (1995, supra, ch. 10 and 16).
Purifted BMDSP obtained
by these methods can be used directly in the following activity assay.
X. Demonstration of BMDSP Activity
An assay for BMDSP-1 activity measures the precipitation of antigen from serum
using
the quantitative precipitin reactiion (Golub, E.S. et al. (1987)Immunology: A
Synthesis, Sinauer
Associates, Sunderland MA, pp. I 13-115). BMDSP-1 is isotopically labeled
using methods
known in the art. Various serum concentrations are added to constant amounts
of labeled
BMDSP-1. BMDSP-1/antigen complexes precipitate out of solution and are
collected by
centrifugation. The amount of precipitable BMDSP-1/antigen complex is
proportional to the
amount of radioisotope detected in the precipitate. The amount of precipitable
BMDSP-1/antigen
complex is plotted against the serum concentration. For various serum
concentrations, a
characteristic precipitin curve is. obtained, in which the amount of
precipitable BMDSP-1/antigen
complex initially increases proportionately with increasing serum
concentration, peaks at the
equivalence point, and then decreases proportionately with further increases
in serum
concentration. Thus, the amount of precipitable BMDSP-1/antigen complex is a
measure of
BMDSP-I activity and is characterized by sensitivity to both limiting and
excess quantities of
antigen.
An assay for BMDSP-2 activity measures the ability of BMDSP-2 to complex with
proteins from bone marrow stromal cells. BMDSP-2, or biologically active
fragments thereof, are
labeled with 'ZSI Bolton-Hunter reagent. (See, e.g., Bolton, A.E. and W.M.
Hunter (1973)
Biochem. J. 133:529-539.) Stromal cell-specific proteins previously arrayed in
the wells of a
microtiter plate are incubated with the labeled BMDSP-2 and washed. The amount
of labeled
BMDSP-2 in each well is quantified and is proportional to the amount of~BMDSP-
2/stromal cell
protein complex. Data obtained using different concentrations of labeled BMDSP-
2 are used to
47
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
calculate the affinity of BMD;SP-2 for stromal cell proteins.
XI. Functional Assays
BMDSP function is assessed by expressing the sequences encoding BMDSP at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA
expression. Vectors of choice include pCMV SPORT plasmid (Life Technologies)
and pCR3.1
plasmid (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus
promoter. 5-10 ug
of recombinant vector are transiently transfected into a human cell line,
preferably of endothet'ial
or hematopoietic origin, using either liposome formulations or
electroporation. I-2 ~cg of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based technique, is
used to identify
transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for
example, their
apoptotic state. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose
events preceding or coincident: with cell death. These events include changes
in nuclear DNA
content as measured by staining of DNA with propidium iodide; changes in cell
size and
granularity as measured by forward light scatter and 90 degree side light
scatter; down-regulation
of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexiin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of BMDSP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding BMDSP and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved
regions of human immunoglobulin G (IgG). Transfected cells are efficiently
separated from
nontransfected cells using magnetic beads coated with either human IgG or
antibody against CD64
(DYNAL, Lake Success NY). mRNA can be purified from the cells using methods
well known
by those of skill in the art. Expression of mRNA encoding BMDSP and other
genes of interest
can be analyzed by northern analysis or microarray techniques.
48
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
XII. Production of BMDSP Specific Antibodies
BMDSP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see,
e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other
purification techniques,
is used to immunize rabbits and to produce antibodies using standard
protocols.
Alternatively, the BMDSP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a
corresponding
oligopeptide is synthesized and used to raise antibodies by means known to
those of skill in the
art. Methods for selection of appropriate epitopes, such as those near the C-
terminus or in
hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995,
supra, ch. 11.)
Typically, oligopeptides 15 residues in length are synthesized using an ABl
431 A
Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(Sigma-Aldrich,
St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to
increase immunogenicity. (See:, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-ICLH complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide activity by, for exarnple, binding the peptide to plastic,
blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring BMDSP Using Specific Antibodies
Naturally occurring or recombinant BMDSP is substantially purified by
immunoaffinity
chromatography using antibodies specific for BMDSP. An immunoaffinity column
is constructed
by covalently coupling anti-BMDSP antibody to an activated chromatographic
resin, such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing BMDSP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of BMDSP (e.g.,
high ionic
strength buffers in the presence of detergent). The column is eluted under
conditions that disrupt
antibody/BMDSP binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope,
such as urea or thiocyanate ion), and BMDSP is collected.
XIV. Identification of Molecules Which Interact with BMDSP
BMDSP, or biological ly active fragments thereof, are labeled with 'z5I Bolton-
Hunter
reagent (Bolton, supra). Candidate molecules previously arrayed in the wells
of a multi-well plate
are incubated with the labeled BMDSP, washed, and any wells with labeled BMDSP
complex are
assayed. Data obtained using different concentrations of BMDSP are used to
calculate values for
the number, affinity, and association of BMDSP with the candidate molecules.
Various modifications and variations of the described methods and systems of
the
49
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
invention will be apparent to those skilled in the art without depa~ ~ing from
the scope and spirit of
the invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited
to such specific embodiments. Indeed, various modifications of the described
modes for carrying
5 out the invention which are obvious to those skilled in molecular biology or
related fields are
intended to be within the scope of the following claims.
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
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51
CA 02345881 2001-03-30
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52
CA 02345881 2001-03-30
PCT/US99/22908
WO OOI20588
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
CORLEY, Neil C.
GUEGLER, Karl J.
LU, Aina Dyung M.
<120> BONE MARROW-DERIVED SERUM PROTEINS
<130> PF-0609 PCT
<140> To Be Assigned
<141> Herewith
<150> _09/165,621; unassigned
<151> 1998-10-02; 1999-10-02
<160> 5
<170> PERL Program
<210> 1
<211> 234
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 135698CD1
<400> 1
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu
1 5 10 15
Pro Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
35 40 45
Ser Gin Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala
65 70 75
Thr Gly Ile Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
80 85 90
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Val Ala Leu
95 100 105
Tyr Tyr Cys Gln Gln Tyr Phe Thr Thr Pro Tyr Thr Phe Gly Gln
110 115 120
Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
125 130 135
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
140 145 150
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
155 160 165
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
170 175 180
l/5
CA 02345881 2001-03-30
WO 00/20588 PCTNS99/22908
Glu Ser Val Thr Glu Gln A.sp Ser Lys Asp Ser Thr Tyr Ser Leu
185 190 195
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
200 205 210
Val Tyr Ala Cys Glu Val T'hr His Gln Gly Leu Ser Ser Pro Val
215 220 225
Thr Lys Ser Phe Asn Arg Gly Glu Cys
230
<210> 2
<211> 254
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1859631CD1
<400> 2
Met Pro Ala Lys Thr Pro Ile Tyr Leu Lys Ala Ala Asn Asn Lys
1 5 10 15
Lys Gly Lys Lys Phe Lys Leu Arg Asp Ile Leu Ser Pro Asp Met
20 25 30
Ile Ser Pro Pro Leu Gly Asp Phe Arg His Thr Ile His Ile Gly
35 40 45
Lys Glu Gly Gln His Asp V.al Phe Gly Asp Ile Ser Phe Leu Gln
50 55 60
Gly Asn Tyr Glu Leu Leu Pro Gly Asn Gln Glu Lys Ala His Leu
65 70 75
Gly Gln Phe Pro Gly His Asn Glu Phe Phe Arg Ala Asn Ser Thr
80 85 90
Ser Asp Ser Val Phe Thr Glu Thr Pro Ser Pro Val Leu Lys Asn
95 100 105
Ala Ile Ser Leu Pro Thr I:Le Gly Gly Ser Gln Ala Leu Met Leu
110 115 120
Pro Leu Leu Ser Pro Val T)ar Phe Asn Ser Lys Gln Glu Ser Phe
125 130 135
Gly Pro Ala Lys Leu Pro A:rg Leu Ser Cys Glu Pro Val Met Glu
140 145 150
Glu Lys Ala Gln Glu Lys Sf=r Ser Leu Leu Glu Asn Gly Thr Val
155 160 165
His Gln Gly Asp Thr Ser T:rp Gly Ser Ser Gly Ser Ala Ser Gln
170 175 180
Ser Ser Gln Gly Arg Asp Scar His Ser Ser Ser Leu Ser Glu Gln
185 190 195
Tyr Pro Asp Trp Pro Ala G.Lu Asp Met Phe Asp His Pro Thr Pro
200 205 210
Cys Glu Leu Ile Lys Gly Lys Thr Lys Ser Glu Glu Ser Leu Ser
215 ' 220 225
Asp Leu Thr Gly Ser Leu Lea Ser Leu Gln Leu Asp Leu Gly Pro
230 235 240
Ser Leu Leu Asp Glu Val Le:u Asn Val Met Asp Lys Asn Lys
245 250
2/5
CA 02345881 2001-03-30
WO 00/20588 PCT/t3S99/22908
<210> 3
<211> 962
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 135698CB1
<400> 3
tcgagccgat tcggctcgag cggctcgagc tcagttagga cccagaggga accatggaag 60
ccccagctca gcttctcttc ctcctgctac tctggctccc agataccacc ggagaaattg 120
tgttgacaca gtctccagcc accctgtctt tgtctccagg ggaaagagcc accctctcct 180
gcagggccag tcagagtgtt agcagctact tagcctggta ccaacagaaa cctggccagg 240
ctcccaggct cctcatctat gatgcatcca acagggccac tggcatccca cccaggttca 300
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag cccgaagatg 360
tggcacttta ttactgtcag caatatttta ctactccgta cacttttggc caggggacca 420
ggctggagat caaacgaact gtggctgcac catctgtctt catcttcccg ccatctgatg 480
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc tatcccagag 540
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc caggagagtg 600
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg acgctgagca 660
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag ggcctgagct 720
cgcccgtcac aaagagcttc aacaggggag agtgttagag ggagaagtgc ccccacctgc 780
tcctcagttc cagcctgacc ccctcccatc ctttggcctc tgaccctttt tccacagggg 840
acctacccct attgcggtcc tccagctcat ctttcacctc acccccctcc tcctccttgg 900
ctttaattat gctaatgttg gaggagaatg aataaataaa gtgaatcttt gcaaaaaaaa 960
as 962
<210> 4
<211> 2546
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 2533
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 1859631CB1
<400> 4
cggggctagc ccggagaccc ggccaccggc ctggggcgcc ttcacgccgt ctcggagcgg 60
ataatgcggt gagcaggcac cacgccggca gactcggctg gatctgcgca cagcggcagg 120
gattgcgtgc gcccgcggga ggcccggggc agcggctggg atcctcagcg gcggccggtt 180
tgtcctggtt gtggtcaaga ctggatgatg taactggctc tctaggaagc ctcacttggc 240
cgtaacctca ggaaggttct ctttgacccc atctcatttc gaagccactt ctgaagccac 300
ttgagaaaaa tgatgtgaca gttcctatca aaaaggattc agaaacatat accatctgtg 360
aagaaagtgg ccctttctcc cgcttgcaaa atagacattc tcaaattcca aaatgccagc 420
caagacccca atttacctga aag~cagccaa taacaagaaa ggaaagaaat ttaaactgag 480
ggacattctg tctcctgata tga.tcagtcc cccgcttgga gactttcgcc acaccatcca 540
cattggcaaa gagggccagc acg~atgtctt tggagatatt tcctttcttc aagggaacta 600
cgagctttta cctggaaacc agg~agaaagc acacctgggc cagttccctg ggcataatga 660
gttcttccgg gccaacagca cct.cggactc tgtgttcaca gaaacgccct ccccggtgct 720
caaaaatgcc atctccctcc cga.ccattgg aggatcccaa gctctcatgt tgcccttatt 780
gtcaccagtg acatttaatt cca.aacagga gtccttcggg ccagcaaagc tgcccaggct 840
3/5
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
tagctgcgag cccgtcatgg agc~aaaaagc tcaggagaaa agcagtctgt tggagaatgg 900
gacagtccac cagggagaca cct:cgtgggg ctccagcggt tctgcatctc agtccagcca 960
aggcagagac agccactcct ccagcctgtc cgaacagtac cccgactggc cagccgagga 1020
catgtttgac catcccaccc cat:gcgagct catcaaggga aagactaagt cagaggagtc 1080
cctctctgac cttacaggtt cccacctctc cctgcagctt gatcttgggc cctcactttt 1140
ggatgaggtg ctgaatgtaa tggataaaaa taagtaacaa gatgccaact tttttccttt 1200
ggggtaaaag gtacaaaaac aaactaacca cagttgaaga gaagggcttc cggagctgta 1260
tttgcagttt tgtgttgggt ttt:ctaaaat aatattctta caaagtattt ttttacctgt 1320
tatgccctgt ttgcaaaaac aatatagaaa aaaacaacaa agcaaaacct atcttggcaa 1380
aaaaaggaag tgagtcagag cccattttca ggaggcattg gtgatgttcg gctcacatat 1440
tgtttgcaga cacacaagaa atcaggcttg gccaggattg gcactagcta tgaagggctg 1500
agcgagtcac attaaggaac ttc:acggaac tttatagcac tccgacattt tctgagcaag 1560
aggaagtcaa aatttattta aca~cctaagc ctttttgtag actcttttct atatattgct 1620 '
taggctcacc atagcgaatt ctc:cagtgtt aaaacttttc tgttttcaca tttgaacttt 1680
atgggttttg gggattttct tgtagttctt atatatccct atatattata tctatattgc 1740
aaaattttga ctgtcagcta cat.gttggta agacacaggc aaagtattac tgtaactaag 1800
ttatttttaa agttaaaata tat.ttttacg tgcctttggc tttttattgc agagtctaca 1860
ttttatagat tctacatcag atg~ttgtcac ttatttccat tgggattcca ttgtaagctg 1920
tgtatgtgcg tgtttggaaa agt.gtattca tacttagttt ttttttcttc atctgttatc 1980
atacttttaa cagcaaccaa taa.cggattg taaagtgtaa aggcacaggt tactcatgat 2040
gcttctgcag agactgtggg cta.caccaca tatgttattt ggaaatatag gtattttagt 2100
acagtacata cttgcattac ata.ggtactt caagcaacac aataaaaagt aaatgataaa 2160
gtgaacttgc ttgtttatag taa.taaacaa gaccataaga gaataagtat agctagagaa 2220
attgcttctc tgaaatgtac atg~agccctt aaggtaagag atgatttcca tctactctca 2280
ttttgattac ttccttatgg tttgagaggc tagaaactga gcctctctac ttttggaaaa 2340
atgaacatgt gaggtcagat ttt.ttttttt ttttttaagt cagcactgat gccaccctct 2400
cagtggtcat ttctgagcat cttcctgact tgaacacctt ctacagcaaa ctcttgcaag 246.0
tccagtttca tccctgtaag gca.aatgtct tttcacgcag aaagtgccat atagacgaga 2520
taaaggcagc tanaacgagg gca.gta 2546
<210> 5
<211> 391
<212> PRT
<213> Homo sapiens
<300>
<308> GenBank ID No: 8338033
<400> 5
Met Pro Gly Pro Gln Gly Gly Arg Gly Ala Ala Thr Met Ser Leu
1 5 10 15
Gly Lys Leu Ser Pro Val Gly Trp Val Ser Ser Ser Gln Gly Lys
20 25 30
Arg Arg Leu Thr Ala Asp Met Ile Ser His Pro Leu Gly Asp Phe
35 40 45
Arg His Thr Met His Val Gly Arg Gly Gly Asp Val Phe Gly Asp
50 55 60
Thr Ser Phe Leu Ser Asn His Gly Gly Ser Ser Gly Ser Thr His
65 70 75
Arg Ser Pro Arg Ser Phe Leu Ala Lys Lys Leu Gln Leu Val Arg
80 85 90
Arg Val Gly Ala Pro Pro Arg Arg Met Ala Ser Pro Pro Ala Pro
95 100 105
Ser Pro Ala Pro Pro Ala Ile Ser Pro Ile Ile Lys Asn Ala Ile
110 115 120
4/5
CA 02345881 2001-03-30
WO 00/20588 PCT/US99/22908
Ser Leu Pro Gln Leu Asn G:Ln Ala Ala Tyr Asp Ser Leu Jal Val
125 130 135
Gly Lys Leu Ser Phe Asp Seer Ser Pro Thr Ser Ser Thr Asp Gly
140 145 150
His Ser Ser Tyr Gly Leu Asp Ser Gly Phe Cys Thr Ile Ser Arg
155 160 165
Leu Pro Arg Ser Glu Lys Pro His Asp Arg Asp Arg Asp Gly Ser
170 175 180
Phe Pro Ser Glu Pro Gly Leu Arg Arg Ser Asp Ser Leu Leu Ser
185 190 195
Phe Arg Leu Asp Leu Asp Le:u Gly Pro Ser Leu Leu Ser Glu Leu
200 205 210
Leu Gly Val Met Ser Leu Pro Glu Ala Pro Ala Ala Glu Thr Pro
215 220 225
Ala Pro Ala Ala Asn Pro Pro Ala Pro Thr Ala Asn Pro Thr Gly
230 235 240
Pro Ala Ala Asn Pro Pro Al.a Thr Thr Ala Asn Pro Pro Ala Pro
245 250 255
Ala Ala Asn Pro Ser Ala Pro Ala Ala Thr Pro Thr Gly Pro Ala
260 265 270
Ala Asn Pro Pro Ala Pro Al.a Ala Ser Ser Thr Pro His Gly His
275 280 285
Cys Pro Asn Gly Val Thr Al.a Gly Leu Gly Pro Val Ala Glu Val
290 295 300
Lys Ser Ser Pro Val Gly Gly Gly Pro Arg Gly Pro Ala Gly Pro
305 310 315
Ala Leu Gly Arg His Trp Gly Ala Gly Trp Asp Gly Gly His His
320 325 330
Tyr Pro Glu Met Asp Ala Arg Gln Glu Arg Val Glu Val Leu Pro
335 340 345
Gln Ala Arg Ala Ser Trp Glu Ser Leu Asp Glu Glu Trp Arg Ala
350 355 360
Pro Gln Ala Gly Ser Arg Thr Pro Val Pro Ser Thr Val Gln Ala
365 370 375
Asn Thr Phe Glu Phe Ala Asp Ala Glu Glu Asp Asp Glu Val Lys
380 385 390
Val
5/5
