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METHODS AND MATERIALS RELATING TO
NOVEI: VON WILLEBRAND/THROMBOSPONDIN-LIKE POLYPEPTIDES
AND POLYNUCLEOTIDES
1. TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by
such
polynucleotides, along with uses for these polynucleotides and proteins, for
example in
therapeutic, diagnostic and research methods. In particular, the invention
relates to a novel
human von Willebrand/Thrombospondin-like polypeptides.
2. BACKGROUND ART
Technolbgy aimed at the discovery of protein factors (including e.g.,
cytokines, such as
lymphokines, interferons, CSFs, chemokines, and interleukins) has matured
rapidly over the
past decade. The now routine hybridization cloning and expression cloning
techniques clone
novel polynucleotides "directly" in the sense that they rely on information
directly related to
the discovered protein (i.e., partial DNA/amino acid sequence of the protein
in the case of
hybridization cloning; activity of the protein in the case of expression
cloning). More recent
"indirect" cloning techniques such as signal sequence cloning, which isolates
DNA sequences
based on the presence of a now well-recognized secretory leader sequence
motif, as well as
various PCR-based or low stringency hybridization-based cloning techniques,
have advanced
the state of the art by making available large numbers of DNA/amino acid
sequences for
proteins that are known to have biological activity, for example, by virtue of
their secreted
nature in the case of leader sequence cloning, by virtue of their cell or
tissue source in the case
of PCR-based techniques, or by virtue of structural similarity to other genes
of known
biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications
in, fox
example, diagnostics, forensics, gene mapping; identification of mutations
responsible for
genetic disorders or other traits, to assess biodiversity, and to produce many
other types of
data and products dependent on DNA and amino acid sequences. It is to these
polypeptides
and the polynucleotides encoding them that the present invention is directed.
In particular, this
invention is directed to a novel von Willebrand/Thrombospondin-like
polypeptides and
polynucleotides.
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3. SUMMARY OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides,
novel
isolated polynucleotides encoding such polypeptides, including recombinant DNA
molecules,
cloned genes or degenerate variants thereof, especially naturally occurring
variants such as
allelic variants, antisense polynucleotide molecules, and antibodies that
specifically recognize
one or more epitopes present on such polypeptides, as well as hybridomas
producing such
antibodies. Specifically, the polynucleotides of the present invention are
based on
polynueleotide isolated from cDNA library prepared from human fetal brain
(Hyseq clone
identification number 15258924).
The compositions of the present invention additionally include vectors,
including
expression vectors, containing the polynucleotides of the invention, cells
genetically engineered
to contain such polynucleotides and cells genetically engineered to express
such
polynucleotides.
The isolated polynucleotides of the invention include, but are not limited to,
a
polynucleotide comprising any one of the nucleotide sequences set forth in the
SEQ ID NQ 1-3;
a polynucleotide comprising any of the full length protein coding sequences of
the SEQ ID NO:
1-3; and a polynucleotide comprising any of the nucleotide sequences of the
mature protein
coding sequences of the SEQ ID NO: 1-3. The polynucleotides of the present
invention also
include, but are not limited to, a polynucleotide that hybridizes under
stringent hybridization
conditions to (a) the complement of any one of the nucleotide sequences set
forth in the SEQ ID
NO: 1-3; (b) a nucleotide sequence encoding SEQ ID NO: 4-7; a polynucleotide
which is an
allelic variant of any polynucleotides recited above; a polynucleotide which
encodes a species
homolog (e.g. orthologs) of any of the proteins recited above; or a
polynucleotide that encodes a
polypeptide comprising a specific domain or truncation of any of the
polypeptides comprising
SEQ ID NO: 4-7.
The nucleic acid sequences of the present invention also include the sequence
information
from the nucleic acid sequences of SEQ ID NO: 1-3. The sequence information
can be a
segment of any one of SEQ ID NO: 1-3 that uniquely identifies or represents
the sequence
information of SEQ ID NO: 1-3. One such segment can be a twenty-mer nucleic
acid sequence
because the probability that a twenty-mer is fully matched in the human genome
is 1 in 300. In
the human genome, there are three billion base pairs in one set of
chromosomes. Because 4°
possible twenty-mers exist, there are 300 times more twenty-mers than there
are base pairs in a
set of human chromosome. Using the same analysis, the probability for a
seventeen-mer to be
fully matched in the human genome is approximately 1 in 5. When these segments
are used in
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arrays for expression studies, fifteen-mer segment can be used. The
probability that the fifteen-
mer is fully matched in the expressed sequences is also approximately one in
five because
expressed sequences comprise less than approximately 5 % of the entire genome
sequence.
Preferably, the nucleic acid fragment or subsequence comprise SEQ ID NO: 1.
S Similarly, when using a sequence information for detecting a single
mismatch, a segment
can be a twenty-five-mer. The probability that the twenty-five mer would
appear in a human
genome with a single mismatch is calculated by multiplying the probability for
a full match
(1-4z5) times the increased probability for mismatch at each nucleotide
position (3 x 25). The
probability that an eighteen mer with a single mismatch can be detected in an
array for expression
studies is approximately one in five. The probability that a twenty-mer with a
single mismatch
can be detected in a human genome is approximately one in five.
A collection as used in this application can be a collection of only one
polynucleotide.
The collection of sequence information or unique identifying information of
each sequence can be
provided on a nucleic acid array. In one embodiment, segments of sequence
information is
provided on a nucleic acid array to detect the polynucleotide that contains
the segment. The array
can be designed to detect full-match or mismatch to the polynucleotide that
contains the segment.
The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the
nucleic acid
sequences recited above; cloning or expression vectors containing the nucleic
acid sequences; and
host cells or organisms transformed with these expression vectors.
Human von WillebrandlThrombospondin-like ("Thrombospondin-Like") polypeptide
(SEQ ID NO: 4) is an approximately 235-amino acid protein with a predicted
molecular mass
of approximately 26 kDa unglycosylated. Protein database searches with the PSI-
BLAST
algorithm (Altschul S.F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402,
herein
2S incorporated by reference) and eMatrix software indicate that SEQ ID NO: 4
is homologous to
african clawed frog thrornbospondin 1 precursor and von Willebrand factor.
Figure 1 shows
the PSI-BLAST amino acid sequence alignment between SEQ ID NO: 4 (also
identified as
"Thrombospondin-Like") and african clawed frog thrombospondin 1 precursor
protein ("Frog
Thrombospondin 1 Precursor"), indicating that the two sequences share 48 %
similarity over 58
amino acid residues of SEQ ID NO: 4 and 36% identity over S8 amino acid
residues of SEQ
ID NO: 4. The sequences of the present invention are expected to have von
Willebrand factor
or Thrombospondin activity, as defined below.
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A predicted approximately eighteen residue signal peptide is encoded from
approximately residue 1 to residue 18 of SEQ ID NO: 4. The extracellular
portion is useful on
its own. This can be confirmed by expression in mammalian cells and sequencing
of the
cleaved product. The signal peptide region was predicted using the Kyte-
Doolittle
hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982),
incorporated herein
by reference). One of skill in the art will recognize that the actual cleavage
site may be
different than that predicted by the computer program.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al.
, J.
Comp. Biol., vol. 6, pp. 219-235 (1999), herein incorporated by reference),
von
Willebrand/Thrombospondin-Like is expected to have von Willebrand factor type
D domain at
residues 124 to 134 of SEQ ID NO: 4 and precursor glycoprotein signal cell
signature at
residues 101 to 150 of SEQ ID NO: 4. The domains corresponding to SEQ ID NO: 4
are as
follows wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E= Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine,
M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine,
T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:
von Willebrand factor type D domain (CWCEDGKVTC designated as SEQ ID NO: 6):
p-value of 1.918e-09, PF00094C (identification number correlating to
signature), located at
residues 124-134 of SEQ ID NO: 4; and
Precursor glycoprotein signal cell signature
(SPCWHLGAMHESRSRWTEPGCSQCWCEDGKVTCEKVRCEAACSHPIPSR designated as
SEQ ID NO: 7): p-value of 9.057e-09, PD02576A (identification number
correlating to
signature), located at residues 101 to 150 of SEQ ID NO: 4. '
Precursor glycoprotein signal cell signature and von Willebrand factor type D
domain
situated in the same region of SEQ ID NO: 4 indicates that SEQ ID NO: 4 may be
involved in the
clot formation process.
Von Willebrand/Thrombospondin-like polypeptide has characteristics of members
of the
thrombospondin family as well as of von Willebrand factors. Members of the
thrombospondin
family are able to inhibit angiogenesis, or new blood vessel formation that
aids tumor growth.
(See, e.g., Volpert et al., Biochem Biophys Res Commun, vol 217, pp. 326-32
(1995); Tokunaga
et al., Br. J. Cancer, vol 79, pp. 354-9 (1999), both incorporated herein by
reference).
Researchers have found that down regulation of thrombospondin may be
associated with Kaposi's
sarcoma, a neoplasm often associated with iatrogenic and aquired
immunosuppression.
(Taraboletti et al., J. Pathol. vol. 188, pp. 76-81 (1999), incorporated
herein by reference).
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Other researchers have found that thrombospondin-1 has a carboxy-terminal cell-
binding domain
that is essential for the adhesion of sickle red blood cells under flow
conditions. (Eiillery et al.,
Blood, vol. 94, pp. 302-309 (1999), incorporated herein by reference).
The polypeptides and polynucleotides of the invention can be utilized, for
example, as
part of methods for the prevention andlor treatment of disorders involving
abnormal bleeding,
von Willebrand disease, haemophilia, angiogenesis, sickle cell anemia, cancer,
and AIDS.
von Willebrand disease is a common hereditary bleeding disease affecting
approximately 1 % of
the United States population. It is caused by a deficiency or defect in a
plasma protein critical
to blood clotting.
The isolated polypeptides of the invention include, but are not limited to, a
polypeptide
comprising SEQ ID NO: 4-7; or the corresponding full length or mature protein.
Polypeptides of
the invention also include polypeptides with biological activity that are
encoded by (a) any of the
polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: I-3;
or (b)
polynucleotides that hybridize to the complement of the polynucleotides of (a)
under stringent
hybridization conditions. Biologically or immunologically active variants of
any of the protein
sequences listed as SEQ ID NO: 4-7 and "substantial equivalents" thereof
(e.g., with at least
about 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 98 % or 99 % amino acid
sequence identity) that
preferably retain biological activity are also contemplated. The polypeptides
of the invention may
be wholly or partially chemically synthesized but are preferably produced by
recombinant means
using the genetically engineered cells (e.g. host cells) of the invention.
The invention also provides compositions comprising a polypeptide of the
invention.
Polypeptide compositions of the invention may further comprise an acceptable
carrier, such as
a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also provides host cells transformed or transfected with a
polynucleotide
of the invention.
The invention also relates to methods for producing a polypeptide of the
invention
comprising growing a culture of the host cells of the invention in a suitable
culture medium
under conditions permitting expression of the desired polypeptide, and
purifying the protein
from the culture or from the host cells. Preferred embodiments include those
in which the
protein produced by such process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a
variety of
techniques known to those skilled in the art of molecular biology. These
techniques include
use as hybridization probes, use as oligomers, or primers, for PCR, use in an
array, use in
computer-readable media, use for chromosome and gene mapping, use in the
recombinant
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production of protein, and use in generation of anti-sense DNA or RNA, their
chemical
analogs and the like. For example, when the expression of an mRNA is largely
restricted to a
particular cell or tissue type, polynucleotides of the invention can be used
as hybridization
probes to detect the presence of the particular cell or tissue mRNA in a
sample using, e.g., in
situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as
expressed sequence tags for identifying expressed genes or, as well known in
the art and
exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed
sequence tags for
physical mapping of the human genome.
A polynucleotide according to the invention can be joined to any of a variety
of other
nucleotide sequences by well-established recombinant DNA techniques (see
Sambrook, J., et
al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, NY).
Useful nucleotide sequences for joining to polypeptides include an assortment
of vectors, e.g.,
plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are
well known in
the art. Accordingly, the invention also provides a vector including a
polynucleotide of the
invention and a host cell containing the polynucleotide. In general, the
vector contains an
origin of replication functional in at least one organism, convenient
restriction endonuclease
sites, and a selectable marker for the host cell. Vectors according to the
invention include
expression vectors, replication vectors, probe generation vectors, and
sequencing vectors. A
host cell according to the invention can be a prokaryotic or eukaryotic cell
and can be a
unicellular organism or part of a multicellular organism.
The polypeptides according to the invention can be used in a variety of
conventional
procedures and methods that are currently applied to other proteins. For
example, a
polypeptide of the invention can be used to generate an antibody that
specifically binds the
polypeptide. Such antibodies, particularly monoclonal antibodies, are useful
for detecting or
quantitating the polypeptide in tissue. The polypeptides of the invention can
also be used as
molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical
condition
which comprises the step of administering to a mammalian subject a
therapeutically effective
amount of a composition comprising a protein of the present invention and a
pharmaceutically
acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be
utilized, for
example, as part of methods for the prevention and/or treatment of disorders
involving
aberrant protein expression or biological activity.
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The methods of the invention also provides methods for the treatment of
disorders as
recited herein which may involve the administration of the polynucleotides or
polypeptides of
the invention to individuals exhibiting symptoms or tendencies related to
disorders as recited
herein. In addition, the invention encompasses methods for treating diseases
ox disorders as
recited herein comprising the step of administering compounds and other
substances that
modulate the overall activity of the target gene products. Compounds and other
substances can
effect such modulation either on the level of target genelprotein expression
or target protein
a
activity. Specifically, methods are provided for preventing, treating or
ameliorating a medical
condition, including neurological diseases, which comprises administering to a
mammalian
subject, including but not limited to humans, a therapeutically effective
amount of a
composition comprising a polypeptide of the invention or a therapeutically
effective amount of
a composition comprising a binding partner of (e.g., antibody specifically
reactive for) von
Willebrand/Thrombospondin-like polypeptides of the invention. The mechanics of
the
particular condition or pathology will dictate whether the polypeptides of the
invention ox
binding partners (or inhibitors) of these would be beneficial to the
individual in need of
treatment.
According to this method, polypeptides of the invention can be administered to
produce
an ita vitro or in vivo inhibition of cellular function. A polypeptide of the
invention can be
administered in viva as a von Willebrand/Thrombospondin-like protein alone or
as an adjunct
to other therapies. Conversely, protein or other active ingredient of the
present invention may
be included in formulations of the particular clotting factor, cytokine,
lymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-
inflammatory agent to
minimize side effects of the clotting factor, cytokine, lymphokine, other
hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
The invention further provides methods for manufacturing medicaments' useful
in the
above described methods.
The present invention further relates to methods for detecting the presence of
the
polynucleotides or polypeptides of the invention in a sample (e.g., tissue or
sample). Such
methods can, for example, be utilized as part of prognostic and diagnostic
evaluation of
disorders as recited herein and for the identification of subjects exhibiting
a predisposition to
such conditions. The invention also provides kits comprising polynucleotide
probes and/or
monoclonal antibodies, and optionally quantitative standards, for carrying out
methods of the
invention. Furthermore, the invention provides methods for evaluating the
efficacy of drugs,
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and monitoring the progress of patients, involved in clinical trials for the
treatment of disorders
as recited above.
The invention also provides methods for the identification of compounds that
modulate
(i.e., increase or decrease) the expression or activity of the polynucleotides
and/or polypeptides
of the invention. Such methods can be utilized, for example, for the
identification of
compounds that can ameliorate symptoms of disorders as recited herein. Such
methods can
include, but are not limited to, assays for identifying compounds and other
substances that
interact with (e.g., bind to) the polypeptides of the invention.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the PSI-BLAST amino acid sequence alignment between SEQ ID NO:
4
(also identified as "Thrombospondin-Like") and african clawed frog
thrombospondin 1
precursor protein ("Frog Thrombospondin 1 Precursor"), indicating that the two
sequences
share 48 % similarity over 58 amino acid residues of SEQ ID NO: 4 and 36 %
identity over 58
IS amino acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine,
D=Aspartic Acid,
E= Glutarnic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,
I~=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine,
S=Serine, T=Threonine, V=Valine, W=Tryptophan,, Y=Tyrosine. Gaps are presented
as
dashes.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 DEFINITIONS
The term "primordial germ cells (PGCs)" refers to a small population of cells
set aside
from other cell lineages particularly from the yolk sac, mesenteries, or
gonadal ridges during
embryogenesis that have the potential to differentiate in to germ cells and
other cells. PGCs
are the source from which GSCs and ES cells are derived
The term "germ line stem cells (GSCs)" refers to stem cells derived from
primordial
stem cells that provide a steady and continuous source of germ cells for the
production of
gametes .
The term "embryonic stem cells (ES)" refers to a cell which can give rise to
many
differentiated cell types in an embryo or an adult, including the germ cells.
The PGCs, the GSCs and the ES cells are capable of self renewal. Thus these
cells not only
populate the germ line and give rise to a plurality of terminally
differentiated cells which
comprise the adult specialized organs, but are able to regenerate themselves.
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The term "totipotent" refers to the capability of a cell to differentiate into
all of the cell
types of an adult organism.
The term "pluripotent" refers to the capability of a cell to differentiate
into a number of
differentiated cell types that are present in an adult organism. A pluripotent
cell is restricted in
its differentiation capability in comparison to a totipotent cell.
The term "nucleotide sequence" refers to a heteropolymer of nucleotides or the
sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide"
are also used
interchangeably herein to refer to a heteropolymer of nucleotides. Generally,
nucleic acid
segments provided by this invention may be assembled from fragments of the
genome and
short oligonucleotide linkers, or from a series of oligonucleotides, or from
individual
nucleotides, to provide a synthetic nucleic acid which is capable of being
expressed in a
recombinant transcriptional unit comprising regulatory elements derived from a
microbial or
viral operon, or a eukaryotic gene.
The terms "oligonucleotide fragment" or a "polynucleotide fragment",
"portion," or
"segment" is a sequence of nucleotide residues which is long enough to use in
polymerase
chain reaction (PCR) or various hybridization procedures to identify or
amplify identical or
related parts of mRNA or DNA molecules. A fragment or segment may uniquely
identify each
polynucleotide sequence of the present invention.
The terms "oligonucleotides" or "nucleic acid probes" are prepared based on
the
polynucleotide sequences provided in the present invention. Oligonucleotides
comprise
portions of such a polynucleotide sequence having at least about 15
nucleotides and usually at
least about 20 nucleotides. Nucleic acid probes comprise portions of such a
polynucleotide
sequence having fewer nucleotides than about 6 kb, usually fewer than about 1
kb. After
appropriate testing to eliminate false positives, these probes may, for
example, be used to
determine whether specific mRNA molecules are present in a cell or tissue or
to isolate similar
nucleic acid sequences from chromosomal DNA as described by Walsh et aI.
(Walsh, P.S. et
al., 1992, PCR Methods Appl 1:241-250).
The term "probes" includes naturally occurring or recombinant or chemically
synthesized single- or double-stranded nucleic acids. They may be labeled by
nick translation,
Klenow fill-in reaction, PCR, or other methods well known in the art. Probes
of the present
invention, their preparation andlor labeling are elaborated in Sambrook, J. et
al., 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or
Ausubel,
F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,
New York
NY, both of which are incorporated herein by reference in their entirety.
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The term "stringent" is used to refer to conditions that are commonly
understood in the
art as stringent. Stringent conditions can include highly stringent conditions
(i.e.,
hybridization to filter-bound DNA in 0.5 M NaHPOa, 7% sodium dodecyl sulfate
(SDS), 1
mM EDTA at 65° C, and washing in O.1X SSC/0.1 % SDS at 68° C),
and moderately stringent
5 conditions (i.e., washing in 0.2X SSC/0.1 %o SDS at 42° C). Other
exemplary hybridization
conditions are described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary
stringent
hybridization conditions include washing in 6X SSC10.05 % sodium pyrophosphate
at 37° C
(for 14-base oligos), 48° C (for 17-base oligos), 55° C (for 20-
base oligos), and 60° C (for
10 23-base oligos).
The term "recombinant," when used herein to refer to a polypeptide or protein,
means
that a polypeptide or protein is derived from recombinant (e.g., microbial,
insect, or
mammalian) expression systems. "Microbial" refers to recombinant polypeptides
or proteins
made in bacterial or fungal (e.g., yeast) expression systems. As a product,
"recombinant
microbial" defines a polypeptide or protein essentially free of native
endogenous substances
and unaccompanied by associated native glycosylation. Polypeptides or proteins
expressed in
most bacterial cultures, e.g., E. coli, will be free of glycosylation
modifications; polypeptides
or proteins expressed in yeast will have a glycosylation pattern in general
different from those
expressed in mammalian cells.
The term "recombinant expression vehicle or vector" refers to a plasmid or
phage or
virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An
expression
vehicle can comprise a transcriptional unit comprising an assembly of (1) a
genetic element or
elements having a regulatory role in gene expression, for example, promoters
or enhancers, (2)
a structural or coding sequence which is transcribed into mRNA and translated
into protein,
and (3) appropriate transcription initiation and termination sequences.
Structural units intended
for use in yeast or eulearyotic expression systems preferably include a leader
sequence enabling
extracellular secretion of translated protein by a host cell. Alternatively,
where recombinant
protein is expressed without a leader or transport sequence, it may include an
amino terminal
methionine residue. This residue may or may not be subsequently cleaved from
the expressed
recombinant protein to provide a final product.
The term "recombinant expression system" means host cells which have stably
integrated a recombinant transcriptional unit into chromosomal DNA or carry
the recombinant
transcriptional unit extrachromosomally. Recombinant expression systems as
defined herein
will express heterologous polypeptides or proteins upon induction of the
regulatory elements
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linked to the DNA segment or synthetic gene to be expressed. This term also
means host cells
which have stably integrated a recombinant genetic element or elements having
a regulatory
role in gene expression, for example, promoters or enl~zancers. Recombinant
expression
systems as defined herein will express polypeptides or proteins endogenous to
the cell upon
induction of the regulatory elements linked to the endogenous DNA segment or
gene to be
expressed. The cells can be prokaryotic or eukaryotic.
The term "open reading frame, " ORF, means a series of nucleotide triplets
coding for
amino acids without any termination codons and is a sequence translatable into
protein.
The term "expression modulating fragment," EMF, means a series of nucleotides
which
modulates the expression of an operably linked ORF or another EMF.
As used herein, a sequence is said to "modulate the expression of an operably
linked
sequence" when the expression of the sequence is altered by the presence of
the EMF. EMFs
include, but are not limited to, promoters, and promoter modulating sequences
(inducible
elements). One class of EMFs are fragments which induce the expression or an
operably
linked ORF in response to a specific regulatory factor or physiological event.
As used herein, an "uptake modulating fragment," UMF, means a series of
nucleotides
which mediate the uptake of a linked DNA fragment into a cell. UMFs can be
readily
identified using known UMFs as a target sequence or target motif with the
computer-based
systems described below.
The presence and activity of a UMF can be confirmed by attaching the suspected
UMF
to a marker sequence. The resulting nucleic acid molecule is then incubated
with an
appropriate host under appropriate conditions and the uptake of the marker
sequence is
determined. As described above, a UMF will increase the frequency of uptake of
a linked
marker sequence.
The term "active" refers to those forms of the polypeptide which retain the
biologic
and/or immunologic activities of any naturally occurring polypeptide.
According to the
invention, the term "biologically active" means that the polypeptide retains
at least one of the
biological activities of the polypeptide of the invention. The term "von
Willebrand factor or
Thrombospondin activity" or "von Willebrand/Thrombospondin activity" or "von
WiIIebrand/Thrombospondin-Iike activity" refers to biological activity that is
similar to the
biological activity of either a von Willebrand factor or a thrombospondin
protein.
The term "naturally occurring polypeptide" refers to polypeptides produced by
cells
that have not been genetically engineered and specifically contemplates
various polypeptides
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arising from post-translational modifications of the polypeptide including,
but not limited to,
acetylation, carboxylation, glycosylation, phosphorylation, lipidation and
acylation.
The term "derivative" refers to polypeptides chemically modified by such
techniques as
ubiquitination, labeling (e.g., with radionuclides or various enzymes),
covalent polymer
attachment such as pegylation (derivatization with polyethylene glycol) and
insertion or
substitution by chemical synthesis of amino acids such as ornithine, which do
not normally
occur in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from
naturally
occurring polypeptides by amino acid insertions, deletions, and substitutions,
created using, a
g. , recombinant DNA techniques. Guidance in determining which amino acid
residues may be
replaced, added or deleted without abolishing activities of interest, may be
found by comparing
the sequence of the particular polypeptide with that of homologous peptides
and minimizing the
number of amino acid sequence changes made in regions of high homology
(conserved regions)
or by replacing amino acids with consensus sequence.
Preferably, amino acid "substitutions" are the result of replacing one amino
acid with
another amino acid having similar structural and/or chemical properties, i. e.
, conservative
amino acid replacements. "Conservative" amino acid substitutions may be made
on the basis
of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity,
andlor the
amphipathic nature of the residues involved. For example, nonpolar
(hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and
methionine; polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids include
arginine, lysine, and
histidine; and negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
"Insertions" or "deletions" are typically in the range of about 1 to 5 amino
acids. The
variation allowed may be experimentally determined by systematically making
insertions,
deletions, or substitutions of amino acids in a polypeptide molecule using
recombinant DNA
techniques and assaying the resulting recombinant variants for activity.
Alternatively, where alteration of function is desired, insertions, deletions
or
non-conservative alterations can be engineered to produce altered
polypeptides. Such
alterations can, for example, alter one or more of the biological functions or
biochemical
characteristics of the polypeptides of the invention. For example, such
alterations may change
polypeptide characteristics such as ligand-binding affinities, interchain
affinities, or
degradation/turnover rate. Further, such alterations can be selected so as to
generate
polypeptides that are better suited for expression, scale up and the like in
the host cells chosen
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for expression. For example, cysteine residues can be deleted or substituted
with another
amino acid residue in order to eliminate disulfide bridges.
As used herein, "substantially equivalent" can refer both to nucleotide and
amino acid
sequences, for example a mutant sequence, that varies from a reference
sequence by one or
more substitutions, deletions, or additions, the net effect of which does not
result in an adverse
functional dissimilarity between the reference and subject sequences.
Typically, such a
substantially equivalent sequence varies from one of those listed herein by no
more than about
20 % (i. e. , the number of individual residue substitutions, additions,
and/or deletions in a
substantially equivalent sequence, as compared to the corresponding reference
sequence,
divided by the total number of residues in the substantially equivalent
sequence is about 0.2 or
less). Such a sequence is said to have 80% sequence identity to the listed
sequence. In one
embodiment, a substantially equivalent, e. g. , mutant, sequence of the
invention varies from a
listed sequence by no more than 10 % (90 % sequence identity); in a variation
of this
embodiment, by no more than 5 % (95 % sequence identity); and in a further
variation of this
embodiment, by no more than 2 % (98 % sequence identity). Substantially
equivalent, e. g. ,
mutant, amino acid sequences according to the invention generally have at
least 95 % sequence
identity with a listed amino acid sequence, whereas substantially equivalent
nucleotide
sequence of the invention can have lower percent sequence identities, taking
into account, for
example, the redundancy or degeneracy of the genetic code. For the purposes of
the present
invention, sequences having substantially equivalent biological activity and
substantially
equivalent expression characteristics are considered substantially equivalent.
For the purposes
of determining equivalence, truncation of the mature sequence (e. g. , via a
mutation which
creates a spurious stop codon) should be disregarded. Sequence identity may be
determined,
e.g., using the Jotun Hein method.
Nucleic acid sequences encoding such substantially equivalent sequences, e. g.
,
sequences of the recited percent identities, can routinely be isolated and
identified via standard
hybridization procedures well known to those of skill in the art.
Where desired, an expression vector may be designed to contain a "signal or
leader
sequence" which will direct the polypeptide through the membrane of a cell.
Such a sequence
may be naturally present on the polypeptides of the present invention or
provided from
heterologous protein sources by recombinant DNA techniques.
A polypeptide "fragment," "portion," ~or "segment" is a stretch of amino acid
residues
of at least about 5 amino acids, often at least about 7 amino acids, typically
at least about 9 to
13 amino acids, and, in various embodiments, at least about 17 or more amino
acids. To be
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14
active, any polypeptide must have sufficient length to display biological
and/or immunological
activity.
Alternatively, recombinant variants encoding these same or similar
polypeptides may be
synthesized or selected by making use of the "redundancy" in the genetic code.
Various codon
substitutions, such as the silent changes which produce various restriction
sites, may be
introduced to optimize cloning into a plasmid or viral vector or expression in
a particular
prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected
in the polypeptide or domains of other peptides added to the polypeptide to
modify the
properties of any part of the polypeptide, to change characteristics such as
ligand-binding
affinities, interchain affinities, or degradation/turnover rate.
The term "activated" cells as used in this application are those which are
engaged in
extracellular or intracellular membrane trafficking, including the export of
secretory or
enzymatic molecules as part of a normal or disease process.
The term "purified" as used herein denotes that the indicated nucleic acid or
polypeptide is present in the substantial absence of other biological
macromolecules, e. g. ,
polynucleotides, proteins, and the like. In one embodiment, the polynucleotide
or polypeptide
is purified such that it constitutes at least 95 % by weight, more preferably
at least 99. 8 % by
weight, of the indicated biological macromolecules present (but water,
buffers, and other small
molecules, especially molecules having a molecular weight of less than 1000
Daltons, can be
present).
The term "isolated" as used herein refers to a nucleic acid or polypeptide
separated
from at least one other component (e.g., nucleic acid or polypeptide) present
with the nucleic
acid or polypeptide in its natural source. In one embodiment, the nucleic acid
or polypeptide is
found in the presence of (if anything) only a solvent, buffer, ion, or other
component normally
present in a solution of the same. The terms "isolated" and "purified" do not
encompass
nucleic acids or polypeptides present in their natural source.
The term "infection" refers to the introduction of nucleic acids into a
suitable host cell
by use of a virus or viral vector.
The term "transformation" means introducing DNA into a suitable host cell so
that the
DNA is replicable, either as an extrachromosomal element, or by chromosomal
integration.
The term "transfection" refers to the taking up of an expression vector by a
suitable
host cell, whether or not any coding sequences are in fact expressed.
The term "intermediate fragment" means a nucleic acid between 5 and 1000 bases
in
length, and preferably between 10 and 40 by in length.
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The term "secreted" includes a protein that is transported across or through a
membrane, including transport as a result of signal sequences in its amino
acid sequence when
it is expressed in a suitable host cell. "Secreted" proteins include without
limitation proteins
secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from
the cell in which
5 they are expressed. "Secreted" proteins also include without limitation
proteins which are
transported across the membrane of the endoplasmic reticulum. "Secreted"
proteins are also
intended to include proteins containing non-typical signal sequences (e.g.
Interleukin-1 Beta,
see Krasney, P.A. and Young, P.R. (1992) Cytokine 4(2):134 -143) and factors
released from
damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W.P. et. al.
(1998) Annu.
10 Rev.Immunol.16:27-SS)
Each of the above terms is meant to encompasses all that is described for
each, unless
the context dictates otherwise.
5.2 NUCLEIC ACIDS AND POLYPEPTIDES OF THE INVENTION
1 S Nucleotide and amino acid sequences of the invention are reported below.
Fragments
of the proteins of the present invention which are capable of exhibiting
biological activity are
also encompassed by the .present invention. Fragments of the protein may be in
linear form or
they may be cyclized using known methods, for example, as described in H. U.
Saragovi, et
al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.
Chem. Soc.
114, 9245-9253 (1992), both of which are incorporated herein by reference.
Such fragments
may be fused to carrier molecules such as immunoglobulins for many purposes,
including
increasing the valency of protein binding sites. For example, fragments of the
protein may be
fused through "linker" sequences to the Fc portion of an immunoglobulin. For a
bivalent form
of the protein, such a fusion could be to the Fc portion of an IgG molecule.
Other
2S immunoglobulin isotypes may also be used to generate such fusions. For
example, a
protein-IgM fusion would generate a decavalent form of the protein of the
invention.
The present invention also provides both full-length and mature forms (for
example,
without a signal sequence or precursor sequence) of the disclosed proteins.
The protein coding
sequence is identified in the sequence listing by translation of the disclosed
nucleotide
sequences. The mature form of such protein may be obtained by expression of a
full-length
polynucleotide in a suitable mammalian cell or other host cell. The sequence
of the mature
form of the protein is also determinable from the amino acid sequence of the
full-length form.
Where proteins of the present invention are membrane bound, soluble forms of
the proteins are
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16
also provided. In such forms, part or aII of the regions causing the proteins
to be membrane
bound are deleted so that the proteins are fully secreted from the cell in
which it is expressed.
The polynucleotides of the invention include naturally occurring or wholly or
partially
synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. SEQ ID NO: 1 -
3
may include all of the coding region of the cDNA or may represent a portion of
the coding
region of the cDNA. Further 5' and 3' sequence can be obtained using methods
known in the
art. For example, full length cDNA or genomic DNA that corresponds to any of
the
polynucleotides of the SEQ ID NO: 1-3 can be obtained by screening appropriate
cDNA or
genomic DNA libraries under suitable hybridization conditions using any of the
polynucleotides of the SEQ ID NO: 1 - 3 or a portion thereof as a probe.
Alternatively, the
polynucleotides of the SEQ ID NO: 1 - 3 may be used as the basis for suitable
primers) that
allow identification andlor amplification of genes in appropriate genomic DNA
or cDNA
libraries.
The nucleic acid sequences of the invention can be assembled ESTs and
sequences
(including cDNA and genomic sequences) obtained from one or more public
databases, such as
dbEST, gbpri, and UniGene. The sequences falling within the scope of the
present invention are
not limited to these specific sequences, but also include allelic and species
variations thereof.
Allelic and species variations can be routinely determined by comparing the
sequence provided in
SEQ ID NO: 1-3, a representative fragment thereof, or a nucleotide sequence at
least 90
identical, preferably 99.9% identical, to SEQ ID NO: 1-3 with a sequence from
another isolate of
the same species. Furthermore, to accommodate codon variability, the invention
includes nucleic
acid.molecules coding for the same amino acid sequences as do the specific
ORFs disclosed
herein. In other words, in the coding region of an ORF, substitution of one
codon foranother
which encodes the same amino acid is expressly contemplated.
Certain nucleic acids of the present invention, designated as SEQ ID NO. 2,
was
assembled using an EST sequence as a seed. The EST sequence can be extended
into a full-
length nucleic acid sequence by programs or algorithms known in the art.
Preferably, a recursive
algorithm is used to extend the seed EST into an extended assemblage, by
pulling additional
sequences from different databases (e.g., Hyseq's database containing EST
sequences, dbEST
version 114, gb pri 114, and UniGene version 101) that belong to this
assemblage. The algorithm
terminates when there was no additional sequences from the databases that will
extend the
assemblage. Further, the inclusion of component sequences into the assemblage
is preferably
based on a BLASTN hit to the extending assemblage with BLAST score greater
than 300 and
percent identity greater than 95 % . BLAST, which stands for Basic Local
Alignment Search
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Tool, is used to search for local sequence alignments (Altschul, S.F., J. Mol.
Evol. 36: 290 - 300
(1993) and Altschul S.F. et al., J. Mol. Biol. 21: 403-10 (1990)). BLAST
produces alignments
of both nucleotide and amino acid sequences to determine sequence similarity.
Because of the
local nature of the alignments, BLAST is especially useful in determining
exact matches.
The EST sequences (SEQ ID NO: 1) can provide identifying sequence information,
representative fragment or segment information, or novel segment information
for the full-length
gene.
The nearest neighbor result for the nucleic acids of the present invention,
including SEQ
ID NO: 2, can be obtained by searching a database using an algorithm or a
program. Preferably,
a FASTA version 3 search against Genpept, using Fastxy algorithm. The nearest
neighbor result
shows the closest homologue for each assemblage from Genpept (and contains the
translated
amino acid sequences for which the assemblage encodes).
The present invention also provides genes corresponding to the cDNA sequences
disclosed
herein. The corresponding genes can be isolated in accordance with known
methods using the
sequence information disclosed herein. Such methods include the preparation of
probes or
primers from the disclosed sequence information for identification and/or
amplification of genes
in appropriate genomic libraries or other sources of genomic materials.
Species homologs (or orthologs) of the disclosed polynucleotides and proteins
axe also
provided by the present invention. Species homologs may be isolated and
identified by making
suitable probes or primers from the sequences provided herein and screening a
suitable nucleic
acid source from the desired species.
The invention also encompasses allelic variants of the disclosed
polynucleotides or
proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide which also
encode proteins which are identical, homologous or related to that encoded by
the
polynucleotides.
5.3 NUCLEIC ACIDS OF THE INVENTION
The isolated polynucleotides of the invention include, but are not limited to,
a
polynucleotide encoding a polypeptide comprising SEQ ID NO: 1-3; or a mature
protein
portion thereof. A preferred nucleic acid sequence is set forth as SEQ ID NO:
3.
The isolated polynucleotides of the invention further include, but are not
limited to a
polynucleotide comprising any of the nucleotide sequence of the SEQ ID NO: 1 -
3; a
polynucleotide comprising the full length protein coding sequence of the
polynucleotides of the
SEQ ID NO: 1 - 3; and a polynucleotide comprising the nucleotide sequence
encoding the
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mature protein coding sequence of the polynucleotides of the SEQ ID NO: 1 - 3.
The
polynucleotides of the present invention also include, but are not limited to,
a polynucleotide
that preferably has von Willebrand/Thrombospondin activity and that hybridizes
under
stringent conditions (a) to the complement of any of the nucleotides sequences
of the SEQ ID
NO: 1 - 3 (b) to a polynucleotide encoding any one of the polypeptides of SEQ
ID NO: 4-7; a
polynucleotide which is an allelic variant of any polynucleotide recited
above; a polynucleotide
which encodes a species homolog of any of the proteins recited above; or a
polynucleotide that
encodes a polypeptide comprising a specific domain or truncation of the
polypeptides of SEQ
ID NO: 4-7. Domains of interest may depend on the nature of the encoded
polypeptide; e.g.,
domains in receptor-like polypeptides include ligand-binding, extracellular,
transmembrane, or
cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like
proteins
include the variable immunoglobulin-like domains; domains in enzyme-like
polypeptides
include catalytic and substrate binding domains; and domains in ligand
polypeptides include
receptor-binding domains.
Polynucleotides encoding preferred polypeptide truncations of the invention
can be used
to generate polynucleotides encoding chimeric or fusion proteins comprising
one or more
domains of the invention and heterologous protein sequences.
The polynucleotides of the invention additionally include the complement of
any of the
polynucleotides recited above.
The polynucleotides of the invention also provide polynucleotides including
nucleotide
sequences that are substantially equivalent to the polynucleotides recited
above.
Polynucleotides according to the invention can have, e. g. , at least about 65
% , at least about
70 % , at least about 75 % , at least about 80 % , more typically at least
about 90 % , and even
more typically at least about 95 % , sequence identity to a polynucleotide
recited above. The
invention also provides the complement of such polynucleotides. The
polynucleotide can be
DNA (genornic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms
for
obtaining such polynucleotides are well known to those of skill in the art and
can include, for
example, methods for determining hybridization conditions which can routinely
isolate
polynucleotides of the desired sequence identities.
A polynucleotide according to the invention can be joined to any of a variety
of other
nucleotide sequences by well-established recombinant DNA techniques (see
Sambrook J et al.
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
NY).
Useful nucleotide sequences for joining to polynucleotides include an
assortment of vectors,
e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like,
that are well
CA 02395770 2002-06-21
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known in the art. Accordingly, the invention also provides a vector including
a polynucleotide
of the invention and a host cell containing the polynucleotide. In general,
the vector contains
an origin of replication functional in at least one organism, convenient
restriction endonuclease
sites, and a selectable marker for the host cell. Vectors according to the
invention include
expression vectors, replication vectors, probe generation vectors, and
sequencing vectors. A
host cell according to the invention can be a prokaryotic or eukaryotic cell
and can be a
unicellular organism or part of a multicellular organism.
The polynucleotides of the present invention also make possible the
development,
through, e.g., homologous recombination or knock out strategies, of animals
that fail to
express functional von Willebrand/Thrombospondin-like polypeptide or that
express a variant
of von Willebrand/Thrombospondin-like polypeptide. Such animals are useful as
models for
studying the in vivo activities of von Willebrand/Thrombospondin-like
polypeptide as well as
for studying modulators of von Willebrand/Thrombospondin-like polypeptide.
In preferred methods to determine biological functions of the polypeptides of
the
I S invention in vivo, one or more genes provided by the invention are either
over expressed ox
inactivated in the germ line of animals using homologous recombination
[Capecchi, Science
244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the
regulatory
control of exogenous or endogenous promoter elements, are known as transgenic
animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination are
referred to as "knockout" animals. Knockout animals, preferably non-human
mammals, can be
prepared as described in U.S. Patent No. 5,557,032, incorporated herein by
reference.
Transgenic animals are useful to determine the roles polypeptides of the
invention play in
biological processes, and preferably in disease states. Transgenic animals are
useful as model
systems to identify compounds that modulate lipid metabolism. Transgenic
animals, preferably
non-human mammals, are produced using methods as described in U.S. Patent No
5,489,743
and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of a polynucleotides of
the
invention promoter is either activated or inactivated to alter the level of
expression of the
polypeptides of the invention. Inactivation can be carried out using
homologous recombination
methods described above. Activation can be achieved by supplementing or even
replacing the
homologous promoter to provide for increased protein expression. The
homologous promoter
can be supplemented by insertion of one or more heterologous enhancer elements
known to
confer promoter activation in a particular tissue.
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Knowledge of von Willebrand/Thrombospondin-like DNA sequences allows for
modification of cells to permit, or increase, expression of endogenous von
Willebrand/Thrombospondin-like polypeptide. Cells can be modified (e.g., by
homologous
recombination) to provide increased von Willebrand/Thrombospondin-like
polypeptide
5 expression by replacing, in whole or in part, the naturally occurring von
Willebrand/Thrombospondin-like promoter with all or part of a heterologous
promoter so that
the cells express von Willebrand/Thrombospondin-like polypeptide at higher
levels. The
heterologous promoter is inserted in such a manner that it is operatively
linked to von
Willebrand/Thrombospondin-like encoding sequences. See, for example, PCT
International
10 Publication No. W094/12650, PCT International Publication No. W092/20808,
and PCT
International Publication No. W091/09955. It is also contemplated that, in
addition to
heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the
multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along
with the
15 heterologous promoter DNA. If linked to the von Willebrand/Thrombospondin-
like coding
sequence, amplification of the marker DNA by standard selection methods
results in co-
amplification of the von Willebrand/Thrombospondin-like coding sequences in
the cells.
The sequences falling within the scope of the present invention are not
limited to the
specific sequences herein described, but also include allelic variations
thereof. Allelic
20 variations can be routinely determined by comparing the nucleotide
sequences provided in the
SEQ ID NO:1 - 3, a representative fragment thereof, or a nucleotide sequence
at least 99.9%
identical to any of the nucleotide sequences of the SEQ ID NO: 1 - 3 with a
sequence from
another isolate of the same species. To accommodate codon variability, the
invention includes
nucleic acid molecules coding for the same amino acid sequences as do the
specific ORFs
disclosed herein. In other words, in the coding region of an ORF, substitution
of one codon
for another which encodes the same amino acid is expressly contemplated. Any
specific
sequence disclosed herein can be readily screened for errors by resequencing a
particular
fragment, such as an ORF, in both directions (i. e. , sequence both strands).
The present invention further provides recombinant constructs comprising a
nucleic
acid having any of the nucleotide sequences of the SEQ ID NO: 1 - 3 or a
fragment thereof or
any other polynucleotides of the invention. In one embodiment, the recombinant
constructs of
the present invention comprise a vector, such as a plasmid or viral vector,
into which a nucleic
acid having any of the nucleotide sequences of the SEQ ID NO: 1 - 3 or a
fragment thereof is
inserted, in a forward or reverse orientation. In the case of a vector
comprising one of the
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ORFs of the present invention, the vector may further comprise regulatory
sequences,
including for example, a promoter, operably linked to the ORF. For vectors
comprising the
EMFs and UMFs of the present invention, the vector may further comprise a
marker sequence
or heterologous ORF operably linked to the EMF or UMF. Large numbers of
suitable vectors
and promoters are known to those of skill in the art and are commercially
available for
generating the recombinant constructs of the present invention. The following
vectors are
provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript
SK, pBs KS,
pNHBa, pNHl6a, pNHlBa, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,
pDR540,
pRITS (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene)
pSVK3, pBPV, pMSG, pSVL (Pharmacia).
The isolated polynucleotide of the invention may be operably linked to an
expression
control sequence such as the pMT2 or pED expression vectors disclosed in
Kaufman et al.,
Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein
recombinantly.
Many suitable expression control sequences are known in the art. General
methods of
I S expressing recombinant proteins are also known and are exemplified in R.
Kaufman, Methods
in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means
that the
isolated polynucleotide of the invention and an expression control sequence
are'~situated within
a vector or cell in such a way that the protein is expressed by a host cell
which has been
transformed (transfected) with the ligated polynucleotide/expression control
sequence.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol
transferase) vectors or other vectors with selectable markers. Two appropriate
vectors are
pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ,
T3, T7, gpt,
lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV
thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-
I. Selection of
the appropriate vector and promoter is well within the level of ordinary skill
in the art.
Generally, recombinant expression vectors will 'include origins of replication
and selectable
markers permitting transformation of the host cell, e. g. , the ampicillin
resistance gene of E.
coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-
expressed gene to
direct transcription of a downstream structural sequence. Such promoters can
be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-
factor, acid
phosphatase, or heat shock proteins, among others. The heterologous structural
sequence is
assembled in appropriate phase with translation initiation and termination
sequences, and
preferably, a leader sequence capable of directing secretion of translated
protein into the
periplasmic space or extracellular medium. Optionally, the heterologous
sequence can encode
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a fusion protein including an amino terminal identification peptide imparting
desired
characteristics, e.g., stabilization or simplified purification of expressed
recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA
sequence encoding a desired protein together with suitable translation
initiation and termination
signals in operable reading phase with a functional promoter. The vector will
comprise one or
more phenotypic selectable markers and an origin of replication to ensure
maintenance of the
vector and to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts
for transformation include E. coli, Bacillus subtilis, Salfnonella
typhirnuriuni and various
species within the genera PseudofnorZas, Streptomyces, and Staphylococcus,
although others
may also be employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for
bacterial
use can comprise a selectable marker and bacterial origin of replication
derived from
commercially available plasmids comprising genetic elements of the well known
cloning vector
pBR322 (ATCC 370I7). Such commercial vectors include, fox example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech,
Madison, WI,
USA). These pBR322 "backbone" sections are combined with an appropriate
promoter and the
structural sequence to be expressed. Following transformation of a suitable
host strain and
growth of the host strain to an appropriate cell density, the selected
promoter is induced or
derepressed by appropriate means (e. g. , temperature shift or chemical
induction) and cells are
cultured for an additional period. Cells are typically harvested by
centrifugation, disrupted by
physical or chemical means, and the resulting crude extract retained for
further purification.
Included within the scope of the nucleic acid sequences of the invention are
nucleic acid
sequence fragments that hybridize under stringent conditions to any of the
nucleotide sequences
of the SEQ ID NO: 1 - 3, or complements thereof, which fragment is greater
than about 10 bp,
preferably 20 to 50 bp, and even greater than 100 bp, greater than 300 bp, or
greater than 500
bp. Fragments of, e.g. 15, 16, or 20 by or more that axe selective for (i.e.
specifically
hybridize to any one of the polynucleotides of the invention) are
contemplated. Probes capable
of specifically hybridizing to a polynucleotide can differentiate
polynucleotide sequences of the
invention from other polynucleotide sequences in the same family of genes or
can differentiate
human genes from genes of other species, and are preferably based on unique
nucleotide
sequences.
In accordance with the invention, polynucleotide sequences comprising the
mature
protein coding sequences corresponding to any one of SEQ ID NO: 4-7, or
functional
equivalents thereof, may be used to generate recombinant DNA molecules that
direct the
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expression of that nucleic acid, or a functional equivalent thereof, in
appropriate host cells.
Also included are the cDNA inserts of any of the clones identified herein.
The nucleic acid sequences of the invention are further directed to sequences
which
encode variants of the described nucleic acids. These amino acid sequence
variants may be
prepared by methods known in the art by introducing appropriate nucleotide
changes into a
native or variant polynucleotide. There are two variables in the construction
of amino acid
sequence variants: the location of the mutation and the nature of the
mutation. Nucleic acids
encoding the amino acid sequence variants are preferably constructed by
mutating the
polynucleotide to encode an amino acid sequence that does not occur in nature.
These nucleic
acid alterations can be made at sites that differ in the nucleic acids from
different species
(variable positions) or in highly conserved regions (constant regions). Sites
at such locations
will typically be modified in series, e.g., by substituting first with
conservative choices (e.g.,
hydrophobic amino acid to a different hydrophobic amino acid) and then with
more distant
choices (e.g., hydrophobic amino acid to a charged amino acid), and then
deletions or
insertions may be made at the target site. Amino acid sequence deletions
generally range from
about 1 to 30 residues, preferably about 1 to 10 residues, and are typically
contiguous. Amino
acid insertions include amino- and/or carboxyl-terminal fusions ranging in
length from one to
one hundred ox more residues, as well as intrasequence insertions of single or
multiple amino
acid residues. Intrasequence insertions may range generally from about 1 to 10
amino
residues, preferably from 1 to 5 residues. Examples of terminal insertions
include the
heterologous signal sequences necessary for secretion or for intracellular
targeting in different
host cells and sequences such as FLAG or poly-histidine sequences useful for
purifying the
expressed protein.
In a preferred method, polynucleotides encoding the novel amino acid sequences
are
changed via site-directed mutagenesis. This method uses oligonucleotide
sequences to alter a
polynucleotide to encode the desired amino acid variant, as well as a
sufficient adjacent
nucleotides on both sides of the changed amino acid to form a stable duplex on
either side of
the site of being changed. In general, the techniques of site-directed
mutagenesis are well
known to those of skill in the art and this technique is exemplified by
publications such as,
Edelman et al., DNA 2:183 (1983). A versatile and efficient method for
producing
site-specific changes in a polynucleotide sequence was published by Zoller and
Smith, Nucleic
Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid
sequence
variants of the novel nucleic acids. When small amounts of template DNA are
used as starting
material, primers) that differs slightly in sequence from the corresponding
region in the
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template DNA can generate the desired amino acid variant. PCR amplification
results in a
population of product DNA fragments that differ from the polynucleotide
template encoding
the polypeptide at the position specified by the primer. The product DNA
fragments replace
the corresponding region in the plasmid and this gives a polynucleotide
encoding the desired
amino acid variant.
A further technique for generating amino acid variants is the cassette
mutagenesis
technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis
techniques
well known in the art, such as, for example, the techniques in Sambrook et
al., supra, and
Current Protocols in Molecular.Biology, Ausubel et al. 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 used in the practice of the invention
for the cloning and
expression of these novel nucleic acids. Such DNA sequences include those
which are capable
of hybridizing to the appropriate novel nucleic acid sequence under stringent
conditions.
Polynucleotides of the invention can also be used to induce immune responses.
For
example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999),
incorporated herein by
reference, nucleic acid sequences encoding a polypeptide may be used to
generate antibodies
against the encoded polypeptide following topical administration of naked
plasmid DNA or
following injection, and preferably intramuscular injection of the DNA. The
nucleic acid
sequences are preferably inserted in a recombinant expression vector and may
be in the form
of naked DNA.
5.4 ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules that
are hybridizable to or complementary to the nucleic acid molecule comprising
the nucleotide
sequence of SEQ ID NO:1-3, or fragments, analogs or derivatives thereof. An
"antisense"
nucleic acid comprises a nucleotide sequence that is complementary to a
"sense" nucleic acid
encoding a protein, e. g. , complementary to the coding strand of a double-
stranded cDNA
molecule or complementary to an mRNA sequence. In specific aspects, antisense
nucleic acid
molecules are provided that comprise a sequence complementary to at least
about 10, 25, 50,
100, 250 or 500 nucleotides or an entire coding strand, or to only a portion
thereof. Nucleic
acid molecules encoding fragments, homologs, derivatives and analogs of a
protein of any of
SEQ ID N0:4-7 or antisense nucleic acids complementary to a nucleic acid
sequence of SEQ
ID NO:1-3 are additionally provided.
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In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding region"
of the coding strand of a nucleotide sequence of the invention. The term
"coding region"
refers to the region of the nucleotide sequence comprising codons which are
translated into
amino acid residues. In another embodiment, the antisense nucleic acid
molecule is antisense to
5 a "noncoding region" of the coding strand of a nucleotide sequence of the
invention. The term
"noncoding region" refers to 5' and 3' sequences which flank the coding region
that are not
translated into amino acids (i. e. , also referred to as 5' and 3'
untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein (e.
g. , SEQ
ID NO: l-3 , antisense nucleic acids of the invention can be designed
according to the rules of
10 Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid
molecule can be
complementary to the entire coding region of a mRNA, but more preferably is an
oligonucleotide that is antisense to only a portion of the coding or noncoding
region of a
mRNA. For example, the antisense oligonucleotide can be complementary to the
region
surrounding the translation start site of a mRNA. An antisense oligonucleotide
can be, for
15 example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in
length. Anantisense
nucleic acid of the invention can be constructed using chemical synthesis or
enzymatic ligation
reactions using procedures known in the art. For example, an antisense nucleic
acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides
or variously modified nucleotides designed to increase the biological
stability of the molecules
20 or to increase the physical stability of the duplex formed between the
antisense and sense
nucleic acids, e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be
used.
Examples of modified nucleotides that can be used to generate the antisense
nucleic acid
include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine,
25 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, S-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, S-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
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antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i. e. , RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a protein according to the invention to thereby inhibit
expression of
the protein, e. g. , by inhibiting transcription and/or translation. The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the case
of an antisense nucleic acid molecule that binds to DNA duplexes, through
specific interactions
in the major groove of the double helix. An example of a route of
administration ofantisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
I S such that they specifically bind to receptors or antigens expressed on a
selected cell surface,
e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens. The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient intracellular
concentrations of
antisense molecules, vector constructs in which the antisense nucleic acid
molecule is placed
under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
-a nomeric nucleic acid molecule. An -anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res
15: 6625-6641). The
antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide
(moue et al.
(1987) Nucleic Acids Res 15 : 6131-6148) or a chimeric RNA -DNA analogue
(Inoue et al.
(1987) FEBS Lett 215: 327-330).
5.5 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of cleaving
a single-stranded nucleic acid, such as a mRNA, to which they have a
complementary region.
Thus, ribozymes (e. g. , hammerhead ribozymes (described in Haselhoff and
Gerlach (1988)
Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to
thereby inhibit
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translation of a mRNA. A ribozyme having specificity for a nucleic acid of the
invention can
be designed based upon the nucleotide sequence of a DNA disclosed herein (i.
e. , SEQ ID
NO: l-3). For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in
which the nucleotide sequence of the active site is complementary to the
nucleotide sequence to
be cleaved in a SECX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.
4,987,071; and
Cech et al. U.S. Pat. No. 5,116,742. Alternatively, SECX mRNA can be used to
select a
catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See,
e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region (e. g. , promoter and/or enhancers) to
form triple
helical structures that prevent transcription of the gene in target cells. See
generally, Helene.
(1991) Anticancer Drug Des.' 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad.
Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of the invention can be modified at
the base
moiety, sugar moiety or phosphate backbone to improve, e. g. , the stability,
hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate backbone of
the nucleic
acids can be modified to generate peptide nucleic acids (see Hyrup et al.
(1996) Bioorg Med
Claeni 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is
replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
standard solid phase peptide synthesis protocols as described in Hyrup et al.
(I996) above;
Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For
example, PNAs can be used as antisense or antigene agents for sequence-
specific modulation
of gene expression by, e. g. , inducing transcription or translation arrest or
inhibiting
replication. PNAs of the invention can also be used, e. g. , in the analysis
of single base pair
mutations in a gene by, e. g. , PNA directed PCR clamping; as artificial
restriction enzymes
when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.
(1996) above); or
as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996),
above;
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e. g. , to
enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
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formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras can be generated that
may
combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e. g. , RNase H and DNA polymerases, to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in terms
of base stacking,
number of bonds between the nucleobases, and orientation (Hyrup {1996) above).
The
synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996)
above and
Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be
synthesized
on a solid support using standard phosphoramidite coupling chemistry, and
modified
nucleoside analogs, e. g. , 5'-(4-rnethoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can
be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl AcidRes
17:
5973-88). PNA monomers are then coupled in a stepwise manner to produce a
chimeric
molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996)
above).
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3' PNA
segment. See, Petersen et al. (1975) Bioorg Med Chern Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e. g. , for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.
U.S.A.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g. , PCT Publication No.
W089/10134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (See,
e. g. , Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents.
(See, e. g. , Zon,
1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, etc.
5.6 HOSTS
The present invention further provides host cells genetically engineered to
contain the
polynucleotides of the invention. For example, such host cells may contain
nucleic acids of the
invention introduced into the host cell using known transformation,
transfection or infection
methods. The present invention still further provides host cells genetically
engineered to
express the polynucleotides of the invention, wherein such polynucleotides are
in operative
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association with a regulatory sequence heterologous to the host cell which
drives expression of
the polynucleotides in the cell.
Knowledge of DNA sequences provided by the invention allows for modification
of
cells to permit, or increase, expression of endogenous polypeptide. Cells can
be modified
(e.g., by homologous recombination) to provide increased polypeptide
expression by replacing,
in whole or in part, the naturally occurring promoter with all or part of a
heterologous
promoter so that the cells express the protein at higher levels. The
heterologous promoter is
inserted in such a manner that it is operatively linked to the desired protein
encoding
sequences. See, for example, PCT International Publication No. WO 94/12650,
PCT
International Publication No. WO 92/20808, and PCT International Publication
No. WO
91/09955. It is also contemplated that, in addition to heterologous promoter
DNA, amplifiable
marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes
carbamyl
phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or
intron DNA may
be inserted along with the heterologous promoter DNA. If linked to the desired
protein coding
sequence, amplification of the marker DNA by standard selection methods
results in
co-amplification of the desired protein coding sequences in the cells.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell,
a lower
eukaryotic host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the host cell
can be effected by
calcium phosphate transfection, DEAF, dextran mediated transfection, or
electroporation
(Davis, L. et al., Basic Methods in Molecular Biology (1986)). The host cells
containing one
of the polynucleotides of the invention, can be used in conventional manners
to produce the
gene product encoded by the isolated fragment (in the case of an ORF) or can
be used to
produce a heterologous protein under the control of the EMF.
Any host/vector system can be used to express one or more of the ORFs of the
present
invention. These include, but are not limited to, eukaryotic hosts such as
HeLa cells, Cv-1
cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as
E. coli and B.
subtilis. The most preferred cells are those which do not normally express the
particular
polypeptide or protein or which expresses the polypeptide or protein at low
natural level.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under the
control of appropriate promoters. Cell-free translation systems can also be
employed to
produce such proteins using RNAs derived from the DNA constructs of the
present invention.
Appropriate cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are
described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual,
Second Edition,
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Cold Spring Harbor, New York (1989), the disclosure of which is hereby
incorporated by
reference.
Various mammalian cell culture systems can also be employed to express
recombinant
protein. Examples of mammalian expression systems include the COS-7 lines of
monkey
5 kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell
lines capable of
expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK
cell lines.
Mammalian expression vectors will comprise an origin of replication, a
suitable promoter and
also any necessary ribosome binding sites, polyadenylation site, splice donor
and acceptor
sites, transcriptional termination sequences, and 5' flanking nontranscribed
sequences. DNA
10 sequences derived from the SV40 viral genome, for example, SV40 origin,
early promoter,
enhancer, splice, and polyadenylation sites may be used to provide the
required nontranscribed
genetic elements. Recombinant polypeptides and proteins produced in bacterial
culture are
usually isolated by initial extraction from cell pellets, followed by one or
more salting-out,
aqueous ion exchange or size exclusion chromatography steps. Protein refolding
steps can be
15 used, as necessary, in completing configuration of the mature protein.
Finally, high
performance liquid chromatography (HPLC) can be employed for final
purification steps.
Microbial cells employed in expression of proteins can be disrupted by any
convenient method,
including freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
A number of types of cells may act as suitable host cells for expression of
the protein.
20 Mammalian host cells include, for example, monkey COS cells, Chinese
Hamster Ovary
(CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Co1o205
cells, 3T3
cells, CV-1 cells, other transformed primate cell lines, normal diploid cells,
cell strains
derived from in vitro culture of primary tissue, primary explants, HeLa cells,
mouse L cells,
BHK, HL-60, U937, HaK or Jurkat cells.
25 Alternatively, it may be possible to produce the protein in lower
eukaryotes such as
yeast, insects or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharonzyces cerevisiae, Schizosaccharornyces pombe, Kluyverornyces strains,
Candida, or
any yeast strain capable of expressing heterologous proteins. Potentially
suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimuriutn,
or any bacterial
30 strain capable of expressing heterologous proteins. If the protein is made
in yeast or bacteria,
it may be necessary to modify the protein produced therein, for example by
phosphorylation or
glycosylation of the appropriate sites, in order to obtain the functional
protein. Such covalent
attachments may be accomplished using known chemical or enzymatic methods.
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In another embodiment of the present invention, cells and tissues may be
engineered to
express an endogenous gene comprising the polynucleotides of the invention
under the control
of inducible regulatory elements, in which case the regulatory sequences of
the endogenous
gene may be replaced by homologous recombination. As described herein, gene
targeting can
be used to replace a gene's existing regulatory region with a regulatory
sequence isolated from
a different gene or a novel regulatory sequence synthesized by genetic
engineering methods.
Such regulatory sequences may be comprised of promoters, enhancers, scaffold-
attachment
regions, negative regulatory elements, transcriptional initiation sites,
regulatory protein binding
sites or combinations of said sequences. Alternatively, sequences which affect
the structure or
stability of the RNA or protein produced may be replaced, removed, added, or
otherwise
modified by targeting. These sequence include polyadenylation signals, mRNA
stability
elements, splice sites, leader sequences for enhancing or modifying transport
or secretion
properties of the protein, or other sequences which alter or improve the
function or stability of
protein or RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the
gene under the control of the new regulatory sequence, e.g., inserting a new
promoter or
enhancer or both upstream of a gene. Alternatively, the targeting event may be
a simple
deletion of a regulatory element, such as the deletion of a tissue-specific
negative regulatory
element. Alternatively, the targeting event may replace an existing element;
for example, a
tissue-specific enhancer can be replaced by an enhancer that has broader or
different cell-type
specificity than the naturally occurring elements. Here, the naturally
occurring sequences are
deleted and new sequences are added. In all cases, the identification of the
targeting event may
be facilitated by the use of one or more selectable marker genes that are
contiguous with the
targeting DNA, allowing for the selection of cells in which the exogenous DNA
has integrated
into the host-cell genome. The identification of the targeting event may also
be facilitated by
the use of one or more marker genes exhibiting the property of negative
selection, such that the
negatively selectable marker is linked to the exogenous DNA, but configured
such that the
negatively selectable marker flanks the targeting sequence, and such that a
correct homologous
recombination event with sequences in the host cell genome does not result in
the stable
integration of the negatively selectable marker. Markers useful for this
purpose include the
Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-
guanine
phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in
accordance with
this aspect of the invention are more particularly described in U.S. Patent
No. 5,272,071 to
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Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International
Application No.
PCT/US92/09627 (W093/09222) by Selden et al.; and International Application
No.
PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated
by
reference herein in its entirety.
5.7 POLYPEPTIDES OF THE INVENTION
The isolated polypeptides of the invention include, but are not limited to, a
polypeptide
comprising: the amino acid sequence set forth as any one of SEQ ID NO: 4-7 or
an amino acid
sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-3 or the
corresponding full length or mature protein. Polypeptides of the invention
also include
polypeptides preferably with biological or immunological activity that are
encoded by: (a) a
polynucleotide having any one of the nucleotide sequences set forth in the SEQ
ID NO: 1-3 or
(b) polynucleotides encoding any one of the amino acid sequences set forth as
SEQ ID NO: 4-7
or (c) polynucleotides that hybridize to the complement of the polynucleotides
of either (a) or
(b) under stringent hybridization conditions. The invention also provides
biologically active or
immunologically active variants of any of the amino acid sequences set forth
as SEQ ID NO:
4-7 or the corresponding full length or mature protein; and "substantial
equivalents" thereof
(e. g. , with at least about 65 % , at least about 70 % , at least about 75 %
, at least about 80 % , at
least about 85 % , at least about 90 % , typically at least about 95 % , more
typically at least about
98%, or most typically at least about 99% amino acid identity) that retain
biological activity.
Polypeptides encoded by allelic variants may have a similar, increased, or
decreased activity
compared to polypeptides comprising SEQ ID NO: 4-7.
Protein compositions of the present invention may further comprise an
acceptable
carrier, such as a hydrophilic, e. g. , pharmaceutically acceptable, carrier.
The invention also relates to methods for producing a polypeptide comprising
growing a
culture of host cells of the invention in a suitable culture medium,
and,purifying the protein
from the cells or the culture in which the cells are grown. For example, the
methods of the
invention include a process for producing a polypeptide in which a host cell
containing a
suitable expression vector that includes a polynucleotide of the invention is
cultured under
conditions that allow expression of the encoded polypeptide. The polypeptide
can be recovered
from the culture, conveniently from the culture medium, or from a lysate
prepared from the
host cells and further purified. Preferred embodiments include those in which
the protein
produced by such process is a full length or mature form of the protein.
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The present invention further provides isolated polypeptides encoded by the
nucleic acid
fragments of the present invention or by degenerate variants of the nucleic
acid fragments of
the present invention. By "degenerate variant" is intended nucleotide
fragments which differ
from a nucleic acid fragment of the present invention (e. g. , an ORF) by
nucleotide sequence
but, due to the degeneracy of the genetic code, encode an identical
polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the ORFs that
encode proteins. A
variety of methodologies known in the art can be utilized to obtain any one of
the isolated
polypeptides or proteins of the present invention. At the simplest level, the
amino acid
sequence can be synthesized using commercially available peptide synthesizers.
This technique
IO is particularly useful in producing small peptides and fragments of larger
polypeptides.
Fragments are useful, for example, in generating antibodies against the native
polypeptide. In
an alternative method, the polypeptide or protein is purified from bacterial
cells which
naturally produce the polypeptide or protein. One skilled in the art can
readily follow known
methods for isolating polypeptides and proteins in order to obtain one of the
isolated
polypeptides or proteins of the present invention. These include, but are not
limited to,
immunochromatography, HPLC, size-exclusion chromatography, ion-exchange
chromatography, and immuno-affinity chromatography. See, e. g. , Scopes,
Protein
Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et
al., in Molecular
Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular
Biology.
Polypeptide fragments that retain biological/immunological activity include
fragments encoding
greater than about 100 amino acids, or greater than about 200 amino acids, and
fragments that
encode specific protein domains.
The polypeptides and proteins of the present invention can alternatively be
purified
from cells which have been altered to express the desired polypeptide or
protein. As used
herein, a cell is said to be altered to express a desired polypeptide or
protein when the cell,
through genetic manipulation, is made to produce a polypeptide or protein
which it normally
does not produce or which the cell normally produces at a lower level. One
skilled in the art
can readily adapt procedures for introducing and expressing either recombinant
or synthetic
sequences into eukaryotic or prokaryotic cells in order to generate a cell
which produces one of
the polypeptides ~or proteins of the present invention.
The purified polypeptides can be used in in vitro binding assays which are
well known
in the art to identify molecules which bind to the polypeptides. These
molecules include but
are not limited to, for e.g., small molecules, molecules from combinatorial
libraries, antibodies
or other proteins. The molecules identified in the binding assay are then
tested for antagonist
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or agonist activity in in vivo tissue culture or animal models that are well
known in the art, In
brief, the molecules are titrated into a plurality of cell cultures or animals
and then tested for
either cell/animal death or prolonged survival of the animal/cells.
In addition, the binding molecules may be complexed with toxins, e.g., ricin
or
cholera, or with other compounds that are toxic to cells. The toxin-binding
molecule complex
is then targeted to a tumor or other cell by the specificity of the binding
molecule for SEQ ID
NO: 4-7.
The protein of the invention may also be expressed as a product of transgenic
animals,
e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep
which are
characterized by somatic or germ cells containing a nucleotide sequence
encoding the protein.
The protein may also be produced by known conventional chemical synthesis.
Methods
for constructing the proteins of the present invention by synthetic means are
known to those
skilled in the art. The synthetically-constructed protein sequences, by virtue
of sharing
primary, secondary or tertiary structural and/or conformational
characteristics with proteins
may possess biological properties in common therewith, including protein
activity. Thus, they
may be employed as biologically active or immunological substitutes for
natural, purified
proteins in screening of therapeutic compounds and in iznmunological processes
for the
development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberately engineered. For example, modifications in the peptide
or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications of
interest in the protein sequences may include the alteration, substitution,
replacement, insertion
or deletion of a selected amino acid residue in the coding sequence. For
example, one or more
of the cysteine residues may be deleted or replaced with another amino acid to
alter the
conformation of the molecule. Techniques for such alteration, substitution,
replacement,
insertion or deletion are well known to those skilled in the art (see, e.g.,
U.S. Pat. No.
4,518,584). Preferably, such alteration, substitution, replacement, insertion
or deletion retains
the desired activity of the protein. Regions of the protein that are important
for the protein
function can be determined by various methods known in the art including the
alanine-scanning
method which involved systematic substitution of single or strings of amino
acids with alanine,
followed by testing the resulting alanine-containing variant for biological
activity. This type of
analysis determines the importance of the substituted amino acids) in
biological activity.
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Other fragments and derivatives of the sequences of proteins which would be
expected
to retain protein activity in whole or in part and are useful for screening or
other
immunological methodologies may also be easily made by those skilled in the
art given the
disclosures herein. Such modifications are encompassed by the present
invention.
5 The protein may also be produced by operably linking the isolated
polynucleotide of the
invention to suitable control sequences in one or more insect expression
vectors, and
employing an insect expression system. Materials and methods for
baculovirus/insect cell
expression systems are commercially available in kit form from, e.g.,
Invitrogen, San Diego,
Calif., U.S.A. (the MaxBatTM kit), and such methods are well known in the art,
as described in
10 Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555
(1957),
incorporated herein by reference. As used herein, an insect cell capable of
expressing a
polynucleotide of the present invention is "transformed. "
The protein of the invention may be prepared by culturing transformed host
cells under
culture conditions suitable to express the recombinant protein. The resulting
expressed protein
15 may then be purified from such culture (i. e. , from culture medium or cell
extracts) using
known purification processes, such as gel filtration and ion exchange
chromatography. The
purification of the protein may also include an affinity column containing
agents which will
bind to the protein; one or more column steps over such affinity resins as
concanavalin
A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseT'"; one or more
steps
20 involving hydrophobic interaction chromatography using such resins as
phenyl ether, butyl
ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form
which will
facilitate purification. For example, it may be expressed as a fusion protein,
such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin
(TRX), or as a
25 His tag. Kits for expression and purification bf such fusion proteins are
commercially
available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway,
N.J.) and
Invitrogen, respectively. The protein can also be tagged with an epitope and
subsequently
purified by using a specific antibody directed to such epitope. One such
epitope ("FLAG ") is
commercially available from Kodak (New Haven, Conn.).
30 Finally, one or more reverse-phase high performance liquid chromatography
(RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant methyl
or other aliphatic groups, can be employed to further purify the protein. Some
or all of the
foregoing purification steps, in various combinations, can also be employed to
provide a
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36
substantially homogeneous isolated recombinant protein. The protein thus
purified is
substantially free of other mammalian proteins and is defined in accordance
with the present
invention as an "isolated protein. "
The polypeptides of the invention include analogs (variants). The polypeptides
of the
invention include von Willebrand/Thrombospondin-like analogs. This embraces
fragments of
von Willebrand/Thrombospondin-like polypeptide of the invention, as well as
von
Willebrand/Thrombospondin-like polypeptide which comprise one or more amino
acids
deleted, inserted, or substituted. Also, analogs of von
Willebrand/Thrombospondin-like
polypeptide of the invention embrace fusions of von Willebrand/Thrombospondin-
like
polypeptide or modifications of von Willebrand/Thrombospondin-like
polypeptide, wherein the
von Willebrand/Thrombospondin-like polypeptide or analog is fused to another
moiety or
moieties, e.g., targeting moiety or another therapeutic agent. Such analogs
may exhibit
improved properties such as activity and/or stability. Examples of moieties
which may be
fused to von Willebrand/Thrombospondin-like polypeptide or an analog include,
for example,
I S targeting moieties which provide for the delivery of polypeptide to
pancreatic cells, e. g. ,
antibodies to pancreatic cells, antibodies to immune cells such as T-cells,
monocytes, dendritic
cells, granulocytes, etc., as well as receptor and ligands expressed on
pancreatic or immune
cells. Other moieties which may be fused to von Willebrand/Thrombospondin-like
polypeptide
include therapeutic agents which are used for treatment, for example,
immunosuppressive
drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.
Also, von
Willebrand/Thrombospondin-like polypeptide may be fused to immunostimulants,
immune
modulators, and other cytokines such as alpha or beta interferon.
5.7.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY
AND SIMILARITY
Preferred identity and/or similarity are designed to give the largest match
between the
sequences tested. Methods to determine identity and similarity are codified in
computer
programs including, but are not limited to, the GCG program package, including
GAP
(Devereux, J., et al., Nucleic Acids Research I2(1):387 (1984); Genetics
Computer Group,
University of Wisconsin, Madison, WI), BLASTP, BLASTN, BLASTX, FASTA
(AItschul,
S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et
al., Nucleic
Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix
software (Wu
et al., J. Comp. Biol., vol. 6, pp. 219-23S (1999), herein incorporated by
reference), eMotif
software (Nevill-Manning et al, ISMB-97, vol 4, pp. 202-209, herein
incorporated by
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37
reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol
Biol, 157, pp.
105-31 (1982), incorporated herein by reference). The BLAST programs are
publicly
available from the National Center for Biotechnology Information (NCBI) and
other sources
(BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, MD 20894; Altschul,
S., et
al., J. Mol. Biol. 215:403-410 (1990).
5.8 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a
"chimeric
protein" or "fusion protein" comprises a polypeptide of the invention
operatively linked to.
another polypeptide. Within a fusion protein the polypeptide according to the
invention can
correspond to all or a portion of a protein according to the invention. In one
embodiment, a
fusion protein comprises at least one biologically active portion of a protein
according to the
invention. In another embodiment, a fusion protein comprises at least two
biologically active
portions of a protein according to the invention. Within the fusion protein,
the term
"operatively linked" is intended to indicate that the polypeptide(s) according
to the invention
and the other polypeptide(s) are fused in-frame to each other. The polypeptide
can be fused to
the N-terminus or C-terminus or in the middle.
For example, in one embodiment a fusion protein comprises a polypeptide
according to
the invention operably linked to the extracellular domain of a second protein.
In another embodiment, the fusion protein is a GST-fusion protein in which the
polypeptide sequences of the invention are fused to the C-terminus of the GST
(i. e. ,
glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein
in
which the polypeptide sequences according to the invention comprise one or
more domains
fused to sequences derived from a member of the immunoglobulin protein family.
The
immunoglobulin fusion proteins of the invention can be incorporated into
pharmaceutical
compositions and administered to a subject to inhibit an interaction between a
ligand and a
protein of the invention on the surface of a cell, to thereby suppress signal
transductionin vivo.
The immunoglobulin fusion proteins can be used to affect the bioavailability
of a cognate
ligand. Inhibition of the ligand/protein interaction may be useful
therapeutically for both the
treatment of proliferative and differentiative disorders, e,g., cancer as well
as modulating
(e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin
fusion proteins of
the invention can be used to bind and to dimerize 2 receptors and thereby
transduce an
intracellular signal. The immunoglobulin fusion proteins may also be used as
immunogens to
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38
produce antibodies in a subject, to purify ligands, and in screening assays to
identify molecules
that inhibit the interaction of a polypeptide of the invention with a ligand.
A chimeric or fusion protein of the invention can be produced by standard
recombinant
DNA techniques. For example, DNA fragments coding for the different
polypeptide
sequences are ligated together in-frame in accordance with conventional
techniques, e. g. , by
employing blunt-ended or stagger-ended termini for ligation, restriction
enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In another
embodiment, the
fusion gene can be synthesized by conventional techniques including automated
DNA
synthesizers. Alternatively, PCR amplification of gene fragments can be
carried out using
anchor primers that give rise to complementary overhangs between two
consecutive gene
fragments that can subsequently be annealed and reamplified to generate a
chimeric gene
sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley ~r Sons, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). A
nucleic acid
encoding a polypeptide of the invention can be cloned into such an expression
vector such that
the fusion moiety is linked in-frame to the protein of the invention.
5.9 GENE THERAPY
Mutations in the polynucleotides of the invention gene may result in loss of
normal
function of the encoded protein. The invention thus provides gene therapy to
restore normal
activity of the polypeptides of the invention; or to treat disease states
involving polypeptides of
the invention. Delivery of a functional genes encoding polypeptides of the
invention to
appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors,
and more particularly
viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or
ex vivo by use of
physical DNA transfer methods (e.g., liposomes or chemical treatments). See,
for example,
Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For
additional reviews
of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989);
Verma, Scientific
American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction
of any one of
the nucleotides of the present invention or a gene encoding the polypeptides
of the present
invention can also be accomplished with extrachromosomal substrates (transient
expression) or
artificial chromosomes (stable expression). Cells may also be cultured ex vivo
in the presence
of proteins of the present invention in order to proliferate or to produce a
desired ,effect on~ or
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activity in such cells. Treated cells can then be introduced in vivo for
therapeutic purposes.
Alternatively, it is contemplated that in other human disease states,
preventing the expression
of or inhibiting the activity of polypeptides of the invention will be useful
in treating the
disease states. It is contemplated that antisense therapy or gene therapy
could be applied to
negatively regulate the expression of polypeptides of the invention.
Other methods inhibiting expression of a protein include the introduction of
antisense
molecules to the nucleic acids of the present invention, their complements, or
their translated
RNA sequences, by methods known in the art, the removal of the nucleic acids
of the present
invention such as using targeted deletion methods, or the insertion of a
negative regulatory
element such as a silencer, which is tissue specific. Further, the
polypeptides of the present
invention can be inhibited by the introduction of antisense molecules that
hybridize to nucleic
acids that encode for the polypeptides of the present invention and by the
removal of a gene that
encode for the polypeptides of the present invention.
The present invention still further provides cells genetically engineered in
vivo to express
the polynucleotides of the invention, wherein such polynucleotides are in
operative association
with a regulatory sequence heterologous to the host cell which drives
expression of the
polynucleotides in the cell. These methods can be used to increase or decrease
the expression of
the polynucleotides of the present invention.
Knowledge of DNA sequences provided by the invention allows for modification
of cells
to permit, increase, or decrease, expression of endogenous polypeptide. Cells
can be modified
(e.g., by homologous recombination) to provide increased polypeptide
expression by replacing, in
whole or in part, the naturally occurring promoter with all or part of a
heterologous promoter so
that the cells express the protein at higher levels. The heterologous promoter
is inserted in such a
manner that it is operatively linked to the desired protein encoding
sequences. See, for example,
PCT International Publication No. WO 94/12650, PCT International Publication
No. WO
92/20808, and PCT International Publication No. WO 91!09955. It is also
contemplated that, in
addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada,
dhfr, and the
multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along
with the
heterologous promoter DNA. If linked to the desired protein coding sequence,
amplification of
the marker DNA by standard selection methods results in co-amplification of
the desired protein
coding sequences in the cells.
In another embodiment of the present invention, cells and tissues may be
engineered to
express an endogenous gene comprising the polynucleotides of the invention
under the control of
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inducible regulatory elements, in which case the regulatory sequences of the
endogenous gene
may be replaced by homologous recombination. As described herein, gene
targeting can be used
to replace a gene's existing regulatory region with a regulatory sequence
isolated from a different
gene or a novel regulatory sequence synthesized by genetic engineering
methods. Such regulatory
5 sequences may be comprised of promoters, enhancers, scaffold-attachment
regions, negative
regulatory elements, transcriptional initiation sites, regulatory protein
binding sites or
combinations of said sequences. Alternatively, sequences which affect the
structure or stability of
the RNA or protein produced may be replaced, removed, added, or otherwise
modified by
targeting. These sequence include polyadenylation signals, mRNA stability
elements, splice sites,
10 leader sequences for enhancing or modifying transport or secretion
properties of the protein, or
other sequences which alter or improve the function or stability of protein or
RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the gene
under the control of the new regulatory sequence, e.g., inserting a new
promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be a simple
deletion of a
15 regulatory element, such as the deletion of a tissue-specific negative
regulatory element.
Alternatively, the targeting event may replace an existing element; for
example, a tissue-specific
enhancer can be replaced by an enhancer that has broader or different cell-
type specificity than the
naturally occurring elements. Here, the naturally occurring sequences are
deleted and new
sequences are added. In aII cases, the identification of the targeting event
may be facilitated by
20 the use of one or more selectable marker genes that are contiguous with the
targeting DNA,
allowing for the selection of cells in which the exogenous DNA has integrated
into the cell
genome. The identification of the targeting event may also be facilitated by
the use of one or
more marker genes exhibiting the property of negative selection, such that the
negatively
selectable marker is linked to the exogenous DNA, but configured such that the
negatively
25 selectable marker flanks the targeting sequence, and such that a correct
homologous
recombination event with sequences in the host cell genome does not result in
the stable
integration of the negatively selectable marker. Markers useful for this
purpose include the
Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-
guanine
phosphoribosyl-transferase (gpt) gene.
30 The gene targeting or gene activation techniques which can be used in
accordance with
this aspect of the invention are more particularly described in U.S. Patent
No. 5,272,071 to
Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International
Application No.
PCT/US92/09627 (W093/09222) by Selden et al.; and International Application
No.
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PCT/US90/06436 (W091/06667) by Skoultchi et al. , each of which is
incorporated by reference
herein in its entirety.
5.10 TRANSGENIC ANIMALS
In preferred methods to determine biological functions of the polypeptides of
the
invention in vivo, one or more genes provided by the invention are either over
expressed or
inactivated in the germ line of animals using homologous recombination
(Capecchi, Science
244:1288-1292 (1989)). Animals in which the gene is over expressed, under the
regulatory
control of exogenous or endogenous promoter elements, are known as transgenic
animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination are
referred to as "knockout" animals. Knockout animals, preferably non-human
mammals, can be
prepared as described in U.S. Patent No. 5,557,032, incorporated herein by
reference.
Transgenic animals are useful to determine the roles polypeptides of the
invention play in
biological processes, and preferably in disease states. Transgenic animals are
useful as model
systems to identify compounds that modulate lipid metabolism. Transgenic
animals, preferably
non-human mammals, are produced using methods as described in U.S. Patent No
5,489,743
and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of a polynucleotides of
the
invention promoter is either activated or inactivated to alter the level of
expression of the
polypeptides of the invention. Inactivation can be carried out using
homologous recombination
methods described above. Activation can be achieved by supplementing or even
replacing the
homologous promoter to provide for increased protein expression. The
homologous promoter
can be supplemented by insertion of one or more heterologous enhancer elements
known to
confer promoter activation in a particular tissue.
2S
5.11 USES AND BIOLOGICAL ACTIVITY OF HUMAN
VON WILLEBRAND/THROMBOSPONDIN-LIKE POLYPEPTIDE
The polynucleotides and proteins of the present invention are expected to
exhibit one or
more of the uses or biological activities (including those associated with
assays cited herein)
identified herein. Uses or activities described for proteins of the present
invention may be
provided by administration or use of such proteins or of polynucleotides
encoding such
proteins (such as, for example, in gene therapies or vectors suitable for
introduction of DNA).
The mechanism underlying the particular condition or pathology will dictate
whether the
polypeptides of the invention, the polynucleotides of the invention or
modulators (activators or
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inhibitors) thereof would be beneficial to the subject in need of treatment.
Thus, "therapeutic
compositions of the invention" include compositions comprising isolated
polynucleotides
(including recombinant DNA molecules, cloned genes and degenerate variants
thereof) or
polypeptides of the invention (including full length protein, mature protein
and truncations or
domains thereof), or compounds and other substances that modulate the overall
activity of the
target gene products, either at the level of target gene/protein expression or
target protein
activity. Such modulators include polypeptides, analogs, (variants), including
fragments and
fusion proteins, antibodies and other binding proteins; chemical compounds
that directly or
indirectly activate or inhibit the polypeptides of the invention (identified,
e.g., via drug
screening assays as described herein); antisense polynucleotides and
polynucleotides suitable
for triple helix formation; and in particular antibodies or other binding
partners that
specifically recognize one or more epitopes of the polypeptides of the
invention.
The polypeptides of the present invention may likewise be involved in cellular
activation or in one of the other physiological pathways described herein.
5.11.1 RESEARCH USES AND UTILITIES
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant
protein for analysis, characterization or therapeutic use; as markers for
tissues in which the
corresponding protein is preferentially expressed (either constitutively or at
a particular stage
of tissue differentiation or development or in disease states); as molecular
weight markers on
gels; as chromosome markers or tags (when labeled) to identify chromosomes or
to map
related gene positions; to compare with endogenous DNA sequences in patients
to identify
potential genetic disorders; as probes to hybridize and thus discover novel,
related DNA
sequences; as a source of information to derive PCR primers for genetic
fingerprinting; as a
probe to "subtract-out" known sequences in the process of discovering other
novel
polynucleotides; for selecting and making oligomers for attachment to a "gene
chip" or other
support, including for examination of expression patterns; to raise anti-
protein antibodies using
DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that described
in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding
the other protein
with which binding occurs or to identify inhibitors of the binding
interaction.
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The polypeptides provided by the present invention can similarly be used in
assays to
determine biological activity, including in a panel of multiple proteins for
high-throughput
screening; to raise antibodies or to elicit another immune response; as a
reagent (including the
labeled reagent) in assays designed to quantitatively determine levels of the
protein (or its
receptor) in biological fluids; as markers for tissues in which the
corresponding polypeptide is
preferentially expressed (either constitutively or at a particular stage of
tissue differentiation or
development or in a disease state); and, of course, to isolate correlative
receptors or ligands.
Where the protein binds or potentially binds to another protein (such as, for
example, in a
receptor-ligand interaction), the protein can be used to identify the other
protein with which
binding occurs or to identify inhibitors of the binding interaction. Proteins
involved in these
binding interactions can also be used to screen for peptide or small molecule
inhibitors or
agonists of the binding interaction.
The polypeptides of the invention are also useful for making antibody
substances that
are specifically immunoreactive with von Willebrand/Thrombospondin-like
proteins.
Antibodies and portions thereof (e.g., Fab fragments) which bind to the
polypeptides of the
invention can be used to identify the presence of such polypeptides in a
sample. Such
determinations are carried out using any suitable immunoassay format, and any
polypeptide of
the invention that is specifically bound by the antibody can be employed as a
positive control.
Any or all of these research utilities are capable of being developed into
reagent grade
or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in the art.
References disclosing such methods include without limitation "Molecular
Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F.
Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to
Molecular
Cloning Techniques", Academic Press, Bergen S. L. and A. R. Kimmel eds., 1987.
5.11.2 NUTRITIONAL USES
Polynucleotides and polypeptides of the present invention can also be used as
nutritional
sources or supplements. Such uses include without limitation use as a protein
or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a
source of carbohydrate.
In such cases the polypeptide or polynucleotide of the invention can be added
to the feed of a
particular organism or can be administered as a separate solid or liquid
preparation, such as in the
form of powder, pills, solutions, suspensions or capsules. In the case of
microorganisms, the
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polypeptide or polynucleotide of the invention can be added to the medium in
or on which the
microorganism is cultured.
Additionally, the polypeptides of the invention can be used as molecular
weight markers,
and as a food supplement. A polypeptide consisting of SEQ ID NO: 5, for
example,has a
molecular mass of approximately 24 kDa in its unprocessed and unglycosylated
state. Protein
food supplements are well known and the formulation of suitable food
supplements including
polypeptides of the invention is within the level of skill in the food
preparation art.
5.11.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION
ACTIVITY
A polypeptide of the present invention may exhibit activity relating to
cytokine, cell
proliferation (either inducing or inhibiting) or cell differentiation (either
inducing or inhibiting)
activity or may induce production of other cytokines in certain cell
populations. A
polynucleotide of the invention can encode a polypeptide exhibiting such
attributes. Many
protein factors discovered to date, including all known cytokines, have
exhibited activity in one
or more factor-dependent cell proliferation assays, and hence the assays serve
as a convenient
confirmation of cytokine activity. The activity of therapeutic compositions of
the present
invention is evidenced by any one of a number of routine factor dependent cell
proliferation
assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9,
B9/11, BaF3,
MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK,
HUVEC, and Caco. Therapeutic compositions of the invention can be used in the
following:
Assays for T-cell or thymocyte proliferation include without limitation those
described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function
3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al. , J. Immunol. 137:3494-
3500, 1986;
Bertagnolli et al., J. Irnmunol. 145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992;
Bowman et al., I.
Immunol. 152:1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node cells or
thymocytes include, without limitation, those described in: Polyclonal T cell
stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E.
e.a. Coligan
eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of
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mouse and human interleukin-y, Schreiber, R. D. In Current Protocols in
Immunology. J. E.
e.a. Coligan eds. Vol I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic cells
include, without limitation, those described in: Measurement of Human and
Murine
5 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P.
E. In Current
Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and
Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau
et al.,
Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938,
1983; Measurement of mouse and human interleukin 6--Nordan, R. In Current
Protocols in
10 Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991;
Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11--Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In
Current Protocols
in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9--Ciarletta, A., Giannotti, J.,
Clark, S. C. and
1S Turner, K. J. In Current Protocols in Immunology. J. E. Coliganeds. Vol 1
pp. 6.13.1, John
Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in: Current
20 Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M.
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter. 3, In
Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their
cellular
receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.
Natl. Acad.
Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-4I1,
1981; Takai et
2S al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-
512, 1988.
5.11.4 STEM CELL GROWTH FACTOR ACTIVITY
A polypeptide of the present invention may exhibit stem cell growth factor
activity and
be involved in the proliferation, differentiation and survival of pluripotent
and totipotent stem
30 cells including primordial germ cells, embryonic stem cells, hematopoietic
stem cells and/or
germ line stem cells. Administration of the polypeptide of the invention to
stem cells in vivo
or ex vivo is expected to maintain and expand cell populations in a
totipotential or
pluripotential state which would be useful for re-engineering damaged or
diseased tissues,
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46
transplantation, manufacture of bio-pharmaceuticals and the development of bio-
sensors. The
ability to produce large quantities of human cells has important working
applications for the
production of human proteins which currently must be obtained from non-human
sources or
donors, implantation of cells to treat diseases such as Parkinson's,
Alzheimer's and other
neurodegenerative diseases; tissues for grafting such as bone marrow, skin,
cartilage, tendons,
bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells,
gastrointestinal
av3
cells and others; and organs for transplantation such as kidney, liver,
pancreas (including islet
cells), heart and lung.
It is contemplated that multiple different exogenous growth factors andlor
cytokines
may be administered in combination with the polypeptide of the invention to
achieve the
desired effect, including any of the growth factors listed herein, other stem
cell maintenance
factors, and specifically including stem cell factor (SCF), leukemia
inhibitory factor (LIF), Flt-
3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor
fused to IL-6,
macrophage inflammatory protein I-alpha (MIP-1-alpha), G-CSF, GM-CSF, thromb
~oietin
(TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural
growth ~,,ctors
and basic fibroblast growth factor (bFGF).
' Since totipotent stem cells can give rise to virtually any mature cell type,
expansion of
c,
these cells in culture will facilitate the production of large quantities of
mature cells.
Techniques for culturing stem cells are known in the art and administration of
polypeptides of
1~,~
the invention, optionally with other growth factors and/or cytokines, is
expected to enhance the
survival and proliferation of the stem cell populations. This can be
accomplished by direct
administration of the polypeptide of the invention to the culture medium.
Alternatively, stroma
cells transfected with a polynucleotide that encodes for the polypeptide of
the invention can
be used as a feeder layer for the stem cell populations in culture or in vivo.
Stromal support
cells for feeder layers may include embryonic bone marrow fibroblasts, bone
marrow stromal
cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Patent
No. 5,690,926).
Stem cells themselves can be transfected with a polynucleotide of the
invention to
induce autocrine expression of the polypeptide of the invention. This will
allow for generation
of undifferentiated totipotential/pluripotential stem cell lines that are
useful as is or that can
then be differentiated into the desired mature cell types. These stable cell
lines can also serve
as a source of undifferentiated totipotential/pluripotential mRNA to create
cDNA libraries and
templates for polymerase chain reaction experiments. These studies would allow
for the
isolation and identification of differentially expressed genes in stem cell
populations that
regulate stem cell proliferation and/or maintenance.
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47
Expansion and maintenance of totipotent stem cell populations will be useful
in the
treatment of many pathological conditions. For example, polypeptides of the
present invention
may be used to manipulate stem cells in culture to give rise to
neuroepithelial cells that can be
used to augment or replace cells damaged by illness, autoimmune disease,
accidental damage
or genetic disorders. The polypeptide of the invention may be useful for
inducing the
proliferation of neural cells and for the regeneration of nerve and brain
tissue, i.e. for the
treatment of central and peripheral nervous system diseases and neuropathies,
as well as
mechanical and traumatic disorders which involve degeneration, death or trauma
to neural cells
or nerve tissue. In addition, the expanded stem cell populations can also be
genetically altered
for gene therapy purposes and to decrease host rejection of replacement
tissues after grafting or
implantation.
Expression of the polypeptide of the invention and its effect on stem cells
can also be
manipulated to achieve controlled differentiation of the stem cells into more
differentiated cell
types. A broadly applicable method of obtaining pure populations of a specific
differentiated
1 S cell type from undifferentiated stem cell populations involves the use of
a cell-type specific
promoter driving a selectable marker. The selectable marker allows only cells
of the desired
type to survive. For example, stem cells can be induced to differentiate into
cardiomyocytes
(Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-
224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue
EfZgineering
eds. Lanza et al., Academic Press (1997)). Alternatively, directed
differentiation of stem cells
can be accomplished by culturing the stem cells in the presence of a
differentiation factor such
as retinoic acid and an antagonist of the polypeptide of the invention which
would inhibit the
effects of endogenous stem cell factor activity and allow differentiation to
proceed.
In vitro cultures of stem cells can be used to determine if the polypeptide of
the
2S invention exhibits stem cell growth factor activity. Stem cells are
isolated from any one of
various cell sources (including hematopoietic stem cells and embryonic stem
cells) and cultured
on a feeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci,
U.S.A., 92: 7844-
7848 (1995), in the presence of the polypeptide of the invention alone ox in
combination with
other growth factors or cytokines. The ability of the polypeptide of the
invention to induce
stem cells proliferation is determined by colony formation on semi-solid
support e.g. as
described by Bernstein et al., Blood, 77: 2316-2321 (1991).
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48
5.11.5 HEMATOPOIESIS REGULATING ACTIVITY
A polypeptide of the present invention may be involved in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell disorders.
Even marginal
biological activity in support of colony forming cells or of factor-dependent
cell lines indicates
involvement in regulating hematopoiesis, e.g. in supporting the growth and
proliferation of
erythroid progenitor cells alone or in combination with other cytokines,
thereby indicating
utility, for example, in treating various anemias or for use in conjunction
with
irradiation/chemotherapy to stimulate the production of erythroid precursors
and/or erythroid
cells; in supporting the growth and proliferation of myeloid cells such as
granulocytes and
monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in
conjunction
with chemotherapy to prevent or treat consequent myelo-suppression; in
supporting the growth
and proliferation of megakaryocytes and consequently of platelets thereby
allowing prevention
or treatment of various platelet disorders such as thrombocytopenia, and
generally for use in
place of or complimentary to platelet transfusions; and/or in supporting the
growth and
proliferation of hematopoietic stem cells which are capable of maturing to any
and all of the
above-mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without limitation,
~aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in
repopulating the stern
cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo
(i.e., in conjunction
with bone marrow transplantation or with peripheral progenitor cell
transplantation
(homologous or heterologous)) as normal cells or genetically manipulated for
gene therapy.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic
lines,
including those assays cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without limitation,
those described in: Johansson et al. Cellular Biology. 15:141-151, 1995;
Keller et al.,
Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,
1993.
Assays for stem cell survival and differentiation (which will identify, among
others,
proteins that regulate lympho-hematopoiesis) include, without limitation,
those described in:
Methylcellulose colony forming assays, Freshney, M. G. In Culture of
Hematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.
1994;
Hirayariia et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic
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49
colony forming cells with high proliferative potential, McNiece, I. K. and
Briddell, R. A. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc.,
New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994;
Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of
Hematopoietic Cells. R.
I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. I994;
Long term
bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter,
M. and Allen, T.
In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-
179, Wiley-Liss,
Inc., New York, N.Y. 1994; Long term culture initiating cell assay,
Sutherland, H. J.,In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162,
Wiley-Liss, Inc.,
New York, N.Y. 1994.
5.11.6 TISSUE GROWTH ACTIVITY
A polypeptide of the present invention also may be involved in bone,
cartilage, tendon,
ligament andlor nerve tissue growth or regeneration, as well as in wound
healing and tissue
repair and replacement, and in healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone
growth in
circumstances where bone is not normally formed, has application in the
healing of bone
fractures and cartilage damage or defects in humans and other animals.
Compositions of a
polypeptide, antibody, binding partner, or other modulator of the invention
may have
prophylactic use in closed as well as open fracture reduction and also in the
improved fixation
of artificial joints. De novo bone formation induced by an osteogenic agent
contributes to the
repair of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and
also is useful in cosmetic plastic surgery.
A polypeptide of this invention may also be involved in attracting bone-
forming cells,
stimulating growth of bone-forming cells, or inducing differentiation of
progenitors of
bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone
degenerative disorders, or
periodontal disease, such as through stimulation of bone and/or cartilage
repair or by blocking
inflammation or processes of tissue destruction (collagenase activity,
osteoclast activity, etc.)
mediated by inflammatory processes may also be possible using the composition
of the
invention.
Another category of tissue regeneration activity that may involve the
polypeptide of the
present invention is tendon/Iigament formation. Induction of tendon/ligament-
like tissue or
other tissue formation in circumstances where such tissue is not normally
formed, has
application in the healing of tendon or ligament tears, deformities and other
tendon or ligament
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defects in humans and other animals. Such a preparation employing a
tendon/ligament-like
tissue inducing protein may have prophylactic use in preventing damage to
tendon or ligament
tissue, as well as use in the improved fixation of tendon or ligament to bone
or other tissues,
and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
S formation induced by a composition of the present invention contributes to
the repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
10 differentiation of progenitors of tendon- or ligament-forming cells, or
induce growth of
tendon/ligament cells or progenitors ex vivo for return ifi vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal tunnel
syndrome and other tendon or ligament defects. The compositions may also
include an
appropriate matrix and/or sequestering agent as a carrier as is well known in
the art.
1 S The compositions of the present invention may also be useful for
proliferation of neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic
disorders, which involve degeneration, death or trauma to neural cells or
nerve tissue. More
specifically, a composition may be used in the treatment of diseases of the
peripheral nervous
20 system, such as peripheral nerve injuries, peripheral neuropathy and
localized neuropathies,
and central nervous system diseases, such as Alzheimer's, Parkinson's disease,
Huntington's
disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further
conditions which
may be treated in accordance with the present invention include mechanical and
traumatic
disorders, such as spinal cord disorders, head trauma and cerebrovascular
diseases such as
25 stroke. Peripheral neuropathies resulting from chemotherapy or other
medical therapies may
also be treatable using a composition of the invention.
Compositions of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
30 Compositions of the present invention may also be involved in the
generation or
regeneration of other tissues, such as organs (including, for example,
pancreas, liver, intestine,
kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including
vascular endothelium) tissue, or for promoting the growth of cells comprising
such tissues.
Part of the desired effects may be by inhibition or modulation of fibrotic
scarring may allow
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51
normal tissue to regenerate. A polypeptide of the present invention may also
exhibit
angiogenic activity.
A composition of the present invention may also be useful for gut protection
or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues, and
conditions resulting from systemic cytokine damage.
A composition of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting the
growth of tissues described above.
Therapeutic compositions of the invention can be used in the following:
9
Assays for tissue generation activity include, without limitation, those
described in:~
International Patent Publication No. W09S/16035 (bone, cartilage, tendon);
International
Patent Publication No. W095/05846 (nerve, neuronal); International Patent
Publication No.
W091/07491 (skin, endothelium).
Assays for wound healing activity include, without limitation, those described
in:
r ~~z
1S Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D.
~~'~:, eds.),
Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest.
Dermatol 71:382-84 (1978).
5.11.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
A polypeptide of the present invention may also exhibit immune stimulating or
immune
suppressing activity, including without limitation the activities for which
assays are,~~iescribed
,..
herein. A polynucleotide of the invention can encode a polypeptide exhibiting
such activities.
A protein may be useful in the treatment of various immune deficiencies and
disorders
(including severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down)
2S growth and proliferation of T and/or B lymphocytes, as well as effecting
the cytolytic activity
of NK cells and other cell populations. These immune deficiencies may be
genetic or be caused
by viral (e.g., HIV) as well as bacterial or fungal infections, or may result
from autoimmune
disorders. More specifically, infectious diseases causes by viral, bacterial,
fungal or other
infection may be treatable using a protein of the present invention, including
infections by
HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria
spp. and
various fungal infections such as candidiasis. Of course, in this regard,
proteins of the present
invention may also be useful where a boost to the immune system generally may
be desirable,
i.e., in the treatment of cancer.
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52
Autoimmune disorders which may be treated using a protein of the present
invention
include, for example, connective tissue disease, multiple sclerosis, systemic
lupus
erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre
syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,
myasthenia gravis,
graft-versus-host disease and autoimmune inflammatory eye disease. Such a
protein (or
antagonists thereof, including antibodies) of the present invention may also
to be useful in the
treatment of allergic reactions and conditions (e.g., anaphylaxis, serum
sickness, drug
reactions, food allergies, insect venom allergies, mastocytosis, allergic
rhinitis,
hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic
dermatitis, allergic contact
dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic
conjunctivitis, atopic
keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary
conjunctivitis and contact
allergies), such as asthma (particularly allergic asthma) or other respiratory
problems. Other
conditions, in which immune suppression is desired (including, for example,
organ
transplantation), may also be treatable using a protein (or antagonists
thereof) of the present
invention. The therapeutic effects of von Willebrand/Thrombospondin-like
polypeptides or
antagonists thereof on allergic reactions can be evaluated by in vivo animals
models such as the
cumulative contact enhancement test (Lastbom et al. , Toxicology 125 : 59-66,
1993), skin prick
test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin
sensitization test (Voter et
al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et
al., J. Toxicol.
Environ. Health 53: 563-79).
Using the proteins of the invention it may also be possible to modulate immune
responses, in a number of ways. Down regulation may be in the form of
inhibiting or blocking
an immune response already in progress or may involve preventing the induction
of an immune .
response. The functions of activated T cells may be inhibited by suppressing T
cell responses
or by inducing specific tolerance in T cells, or both. Immunosuppression of T
cell responses is
generally an active, non-antigen-specific, process which requires continuous
exposure of the T
cells to the suppressive agent. Tolerance, which involves inducing non-
responsiveness or
anergy in T cells, is distinguishable from immunosuppression in that it is
generally
antigen-specific and persists after exposure to the tolerizing agent has
ceased. Operationally,
tolerance can be demonstrated by the lack of a T cell response upon reexposure
to specific
antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as, for example, B7)), e.g.,
preventing high
level lymphokine synthesis by activated T cells, will be useful in situations
of tissue, skin and
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53
organ transplantation and in graft-versus-host disease (GVHD). For example,
blockage of T
cell function should result in reduced tissue destruction in tissue
transplantation. Typically, in
tissue transplants, rejection of the transplant is initiated through its
recognition as foreign by T
cells, followed by an immune reaction that destroys the transplant. The
administration of a
therapeutic composition of the invention may prevent cytokine synthesis by
immune cells, such
as T cells, and thus acts as an immunosuppressant. Moreover, a lack of
costimulation may also
be sufficient to anergize the T cells, thereby inducing tolerance in a
subject. Induction of
long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the
necessity of
repeated administration of these blocking reagents. To achieve sufficient
immunosuppression
or tolerance in a subject, it may also be necessary to block the function of a
combination of B
lymphocyte antigens.
The efficacy of particular therapeutic compositions in preventing organ
transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac grafts
in rats and xenogeneic pancreatic islet cell grafts in mice, both of which
have been used to
examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as
described in
Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA,
89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed.,
Fundamental
Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine
the effect
of therapeutic compositions of the invention on the development of that
disease.
Blocking antigen function may also be therapeutically useful for treating
autoimnnune
diseases. Many autoimmune disorders are the result of inappropriate activation
of T cells that
are reactive against self tissue and which promote the production of cytokines
and
autoantibodies involved in the pathology of the diseases. Preventing the
activation of
autoreactive T cells may reduce or eliminate disease symptoms. Administration
of reagents
which block stimulation of T cells can be used to inhibit T cell activation
and prevent
production of autoantibodies or T cell-derived cytokines which may be involved
in the disease
process. Additionally, blocking reagents may induce antigen-specific tolerance
of autoreactive
T cells which could lead to long-term relief from the disease. The efficacy of
blocking reagents
in preventing or alleviating autoimmune disorders can be determined using a
number of
well-characterized animal models of human autoimmune diseases. Examples
include murine
experimental autoimmune encephalitis, systemic lupus erythmatosis in
MRL/lpr/lpr mice or
NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in
NOD mice and
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54
BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology,
Raven Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (e.g., a B lymphocyte antigen function),
as a means
of up regulating immune responses, may also be useful in therapy. Upregulation
of immune
responses may be in the form of enhancing an existing immune response or
eliciting an initial
immune response. For example, enhancing an immune response may be useful in
cases of viral
infection, including systemic viral diseases such as influenza, the common
cold, and
encephalitis.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient by
removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-pulsed
APCs either expressing a peptide of the present invention or together with a
stimulatory form
of a soluble peptide of the present invention and reintroducing the in vitro
activated T cells into
the patient. Another method of enhancing anti-viral immune responses would be
to isolate
infected cells from a patient, transfect them with a nucleic acid encoding a
protein of the
present invention as described herein such that the cells express all or a
portion of the protein
on their surface, and reintroduce the transfected cells into the patient. The
infected cells would
now be capable of delivering a costimulatory signal to, and thereby activate,
T cells in vivo.
A polypeptide of the present invention may provide the necessary stimulation
signal to
T cells to induce a T cell mediated immune response against the transfected
tumor cells. In
addition, tumor cells which lack MHC class I or MHC class II molecules, or
which fail to
reexpress sufficient mounts of MHC class I or MHC class II molecules, can be
transfected
with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain
truncated portion) of
an MHC class I alpha chain protein and beta sub 2 microglobulin protein or an
MHC class II
alpha chain protein and an MHC class II beta chain protein to thereby express
MHC class I or
MHC class II proteins on the cell surface. Expression of the appropriate class
I or class II
MHC in conjunction with a peptide having the activity of a B lymphocyte
antigen (e.g., B7-1,
B7-2, B7-3) induces a T cell mediated immune response against the transfected
tumor cell.
Optionally, a gene encoding an antisense construct which blocks expression of
an MHC class II
associated protein, such as the invariant chain, can also be cotransfected
with a DNA encoding
a peptide having the activity of a B lymphocyte antigen to promote
presentation of tumor
associated antigens and induce tumor specific immunity. Thus, the induction of
a T cell
mediated immune response in a human subject may be sufficient to overcome
tumor-specific
tolerance in the subject.
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The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek,
S D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function
3.1-3.19;
Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad.
Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et
al., J.
Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986;
Takai et al.,
10 J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
1 S Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which
will identify, among others, proteins that modulate T-cell dependent antibody
responses and
that affect Thl/Th2 profiles) include, without limitation, those described in:
Maliszewski, J.
Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro
antibody production,
Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E, e:a.
Coligan eds.
20 Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins
that generate predominantly Th1 and CTL responses) include, without
limitation, those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M.
Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and
2S Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
3500, 1986;
Takai et al., J-. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783,
1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed
30 by dendritic cells that activate naive T-cells) include, without
limitation, those described in:
Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine
173:549-559, 1991; Macatonia et al., Journal of Immunology 1S4:S071-5079,
1995; Porgador
et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al.,
Journal of Virology
67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al.,
Journal of
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Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of
Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental
Medicine
172.631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins
that prevent apoptosis after superantigen induction and proteins that regulate
lymphocyte
homeostasis) include, without limitation, those described in: Darzynkiewicz et
al., Cytometry
13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al.,
Cancer
Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of
Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993;
Gorczyca et
al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development
include, without limitation, those described in: Antica et al. , Blood 84:111-
117, 1994; Fine et
al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778,
1995; Toki et
al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
5.11.8 ACTIVINIINHIBIN ACTIVITY
A polypeptide of the present invention may also exhibit activin- or inhibin-
related
activities. A polynucleotide of the invention may encode a polypeptide
exhibiting such
characteristics. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to stimulate
the release of follicle stimulating hormone (FSH). Thus, a polypeptide of the
present
invention, alone or in heterodimers with a member of the inhibin family, may
be useful as a
contraceptive based on the ability of inhibins to decrease fertility in female
mammals and
decrease spermatogenesis in male mammals. Administration of sufficient amounts
of other
inhibins can induce infertility in these mammals. Alternatively, the
polypeptide of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin
group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for
advancement
of the onset of fertility in sexually immature mammals, so as to increase the
lifetime
reproductive performance of domestic animals such as, but not limited to,
cows, sheep and
pigs.
The activity of. a polypeptide of the invention may, among other means, be
measured by
the following methods.
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Assays for activin/inhibin activity include, without limitation, those
described in: Vale
et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986;
Vale et al.,
Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et
al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
5.11.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or
chemokinetic
activity for mammalian cells, including, for example, monocytes, fibroblasts,
neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A
polynucleotide of the
invention can encode a polypeptide exhibiting such attributes. Chemotactic and
chemokinetic
receptor activation can be used to mobilize or attract a desired cell
population to a desired site
of action. Chemotactic or chemokinetic compositions (e.g. proteins,
antibodies, binding
partners, or modulators of the invention) provide particular advantages in
treatment of wounds
and other trauma to tissues, as well as in treatment of localized infections.
For example,
attraction of lymphocytes, monocytes or neutrophils to tumors or sites of
infection may result
in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it can
stimulate, directly or indirectly, the directed orientation or movement of
such cell population.
Preferably, the protein or peptide has the ability to directly stimulate
directed movement of
cells. Whether a particular protein has chemotactic activity for a population
of cells can be
readily determined by employing such protein or peptide in any known assay for
cell
chernotaxis.
Therapeutic compositions of the invention can be used in the following:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration of
cells across a membrane as well as the ability of a protein to induce the
adhesion of one cell
population to another cell population. Suitable assays for movement and
adhesion include,
without limitation, those described in: Current Protocols in Immunology, Ed by
J. E. Coligan,
A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene
Publishing
Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines
6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al.
APMIS
103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al.
J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.
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5.11.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
A polypeptide of the invention may also be involved in hemostatis or
thrombolysis or
thrombosis. A polynucleotide of the invention can encode a polypeptide
exhibiting such
attributes. Compositions may be useful in treatment of various coagulation
disorders
(including hereditary disorders, such as hemophilias) or to enhance
coagulation and other
hemostatic events in treating wounds resulting from trauma, surgery or other
causes. A
composition of the invention may also be useful for dissolving or inhibiting
formation of
thromboses and fox treatment and prevention of conditions resulting therefrom
(such as, for
example, infarction of cardiac and central nervous system vessels (e.g.,
stroke).
Therapeutic compositions of the invention can be used in the following:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al. , J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al. , Thrombosis
Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
5.11.11 CANCER DIAGNOSIS AND THERAPY
Polypeptides of the invention may be involved in cancer cell generation,
proliferation or
metastasis. Detection of the presence or amount of polynucleotides or
polypeptides of the
invention may be useful for the diagnosis and/or prognosis of one or more
types of cancer.
For example, the presence or increased expression of a
polynucleotidelpolypeptide of the
invention may indicate a hereditary risk of cancer, a precancerous condition,
or an ongoing
malignancy. Conversely, a defect in the gene or absence of the polypeptide may
be associated
with a cancer condition. Identification of single nucleotide polymorphisms
associated with
cancer or a predisposition to cancer may also be useful for diagnosis or
prognosis.
Cancer treatments promote tumor regression by inhibiting tumor cell
proliferation,
inhibiting angiogenesis (growth of new blood vessels that is necessary to
support tumor
growth) and/or prohibiting metastasis by reducing tumor cell motility or
invasiveness.
Therapeutic compositions of the invention may be effective in adult and
pediatric oncology
including in solid phase turnors/malignancies, locally advanced tumors, human
soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood cell
malignancies including
multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck
cancers
including mouth cancer, larynx cancer and thyroid cancer, lung cancers
including small cell
carcinoma and non-small cell cancers, breast cancers including small cell
carcinoma and ductal
carcinoma, gastrointestinal cancers including esophageal cancer, stomach
cancer, colon cancer,
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colorectal cancer and polyps associated with colorectal neoplasia, pancreatic
cancers, liver
cancer, urologic cancers including bladder cancer and prostate cancer,
malignancies of the
female genital tract including ovarian carcinoma, uterine (including
endometrial) cancers, and
solid tumor in the ovarian follicle, kidney cancers including xenal cell
carcinoma, brain cancers
including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors,
gliomas, metastatic
tumor cell invasion in the central nervous system, bone cancers including
osteomas, skin
cancers including malignant melanoma, tumor progression of human skin
keratinocytes,
squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and
Karposi's sarcoma.
Polypeptides, polynucleotides, or modulators of polypeptides of the invention
(including
inhibitors and stimulators of the biological activity of the polypeptide of
the invention) may be
administered to treat cancer. Therapeutic compositions can be administered in
therapeutically
effective dosages alone or in combination with adjuvant cancer thexapy such as
surgery,
chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide
a beneficial
effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting
metastasis, or
otherwise improving overall clinical condition, without necessarily
eradicating the cancer.
The composition can also be administered in therapeutically effective amounts
as a
portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of
the polypeptide or
modulator of the invention with one or more anti-cancer drugs in addition to a
pharmaceutically acceptable carrier for delivery. The use of anti-cancer
cocktails as a cancer
treatment is routine. Anti-cancer drugs that are well known in the art and can
be used as a
treatment in combination with the polypeptide or modulator of the invention
include:
Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan,
Carboplatin,
Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine
HCl
(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCI,
Doxorubicin HCI,
Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-
Fluorouracil (5-Fu),
Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a,
Interferon
Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,
Mechlorethamine
HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX),
Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI,
Streptozocin,
Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine
sulfate, Amsacrine,
Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin,
Semustine,
Teniposide, and Vindesine sulfate.
In addition, therapeutic compositions of the invention may be used for
prophylactic
treatment of cancer. There are hereditary conditions andlor environmental
situations (e.g.
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exposure to carcinogens) known in the art that predispose an individual to
developing cancers.
Under these circumstances, it may be beneficial to treat these individuals
with therapeutically
effective doses of the polypeptide of the invention to reduce the risk of
developing cancers.
In vitro models can be used to determine the effective doses of the
polypeptide of the
5 invention as a potential cancer treatment. These in vitro models include
proliferation assays of
cultured tumor cells, growth of cultured tumor cells in soft agar (see
Freshney, (1987) Culture
of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18
and Ch 21),
tumor systems in nude mice as described in Giovanella et al., J. Natl. Can.
Inst., 52: 921-30
(1974), mobility and invasive potential of tumor cells in Boyden Chamber
assays as described
10 in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis
assays such as
induction of vascularization of the chick chorioallantoic membrane or
induction of vascular
endothelial cell migration as described in Ribatta et al., Intl. J. Dev.
Biol., 40: 1189-97 (1999)
and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999) respectively. Suitable
tumor cells lines
are available, e.g. from American Type Tissue Culture Collection catalogs.
5.11.12 RECEPTORILIGAND ACTIVITY
A polypeptide of the present invention may also demonstrate activity as
receptor,
receptor ligand or inhibitor or agonist of receptor/ligand interactions. A
polynucleotide of the
invention can encode a polypeptide exhibiting such characteristics. Examples
of such receptors
and ligands include, without limitation, cytokine receptors and their ligands,
receptor kinases
and their ligands, receptor phosphatases and their ligands, receptors involved
in cell-cell
interactions and their ligands (including without limitation, cellular
adhesion molecules (such
as selectins, integrins and their ligands) and receptor/ligand pairs involved
in antigen
presentation, antigen recognition and development of cellular and humoral
immune responses.
Receptors and ligands are also useful for screening of potential peptide or
small molecule
inhibitors of the relevant receptor/ligand interaction. A protein of the
present invention
(including, Without limitation, fragments of receptors and ligands) may
themselves be useful as
inhibitors of receptor/ligand interactions.
The activity of a polypeptide of the invention may, among other means, be
measured by
the following methods:
Suitable assays far receptor-ligand activity include without limitation those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies,
E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-
7.28.22),
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Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J.
Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989;
Stoltenborg et al., J.
Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
By way of example, the polypeptides of the invention may be used as a receptor
for a
ligand(s) thereby transmitting the biological activity of that ligand(s).
Ligands may be
identified through binding assays, affinity chromatography, dihybrid screening
assays, BIAcore
assays, gel overlay assays, or other methods known in the art.
Studies characterizing drugs or proteins as agonist or antagonist or partial
agonists or a
partial antagonist require the use of other proteins as competing ligands. The
polypeptides of
the present invention or ligand(s) thereof may be labeled by being coupled to
radioisotopes,
colorimetric molecules or a toxin molecules by conventional methods. ("Guide
to Protein
Purification" Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990)
Academic
Press, Inc. San Diego). Examples of radioisotopes include, but are not limited
to, tritium and
carbon-14 . Examples of colorimetric molecules include, but are not limited
to, fluorescent
molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
Examples of
toxins include, but are not limited, to ricin.
5.11.13 DRUG SCREENING
This invention is particularly useful for screening chemical compounds by
using the
novel polypeptides or binding fragments thereof in any of a variety of drug
screening
techniques. The polypeptides or fragments employed in such a test may either
be free in
solution, affixed to a solid support, borne on a cell surface or located
intracellularly. One
method of drug screening utilizes eukaryotic or prokaryotic host cells which
are stably
transformed with recombinant nucleic acids expressing the polypeptide or
fragment. Drugs
are screened against such transformed cells in competitive binding assays.
Such cells, either in
viable or fixed form, can be used for standard binding assays. One may
measure, for
example, the formation of complexes between polypeptides of the invention or
fragments and
the agent being tested or examine the diminution in complex formation between
the novel
polypeptides and an appropriate cell line, which are well known in the art.
Sources for test compounds that may be screened for ability to bind to or
modulate
(i.e., increase or decrease) the activity of polypeptides of the invention
include (1) inorganic
and organic chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries
comprised of either random or mimetic peptides, oligonucleotides or organic
molecules.
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Chemical libraries may be readily synthesized or purchased from a number of
commercial sources, and may include structural analogs of known compounds or
compounds
that are identified as "hits" or "leads" via natural product screening.
The sources of natural product libraries are microorganisms (including
bacteria and
fungi), animals, plants or other vegetation, or marine organisms, and
libraries of mixtures for
screening may be created by: (1) fermentation and extraction of broths from
soil, plant or
marine microorganisms or (2) extraction of the organisms themselves. Natural
product
libraries include polyketides, non-ribosomal peptides, and (non-naturally
occurring) variants
thereof. For a review, see Science 282:63-68 (1998).
Combinatorial libraries are composed of large numbers of peptides,
oligonucleotides or
organic compounds and can be readily prepared by traditional automated
synthesis methods,
PCR, cloning or proprietary synthetic methods. Of particular interest are
peptide and
oligonucleotide combinatorial libraries. Still other libraries of interest
include peptide, protein,
peptidomimetic, multiparallel synthetic collection, recombinatorial, and
polypeptide libraries.
For a review of combinatorial chemistry and libraries created therefrom, see
Myers, Curr.
Opita. BiotechrZOl. 8:701-707 (1997). For reviews and examples of
peptidomimetic libraries,
see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr
Opin ClZem
Biol, 1(1):114-19 (1997); Dorner et al., Bioorg Med Chern, 4(5):709-15 (1996)
(alkylated
dipeptides) .
Identification of modulators through use of the various libraries described
herein
permits modification of the candidate "hit" (or "lead") to optimize the
capacity of the "hit" to
bind a polypeptide of the invention. The molecules identified in the binding
assay are then
tested for antagonist or agonist activity in in vivo tissue culture or animal
models that are well
known in the art. In brief, the molecules are titrated into a plurality of
cell cultures or animals
and then tested for either cell/animal death or prolonged survival of the
animallcells.
The binding molecules thus identified may be complexed with toxins, e.g.,
ricin or
cholera, or with other compounds that are toxic to cells such as
radioisotopes. The
toxin-binding molecule complex is then targeted to a tumor or other cell by
the specificity of
the binding molecule for a polypeptide of the invention. Alternatively, the
binding molecules
may be complexed with imaging agents for targeting and imaging purposes.
5.11.14 ASSAY FOR RECEPTOR ACTIVITY
The invention also provides methods to detect specific binding of a
polypeptide e.g. a
ligand or a receptor. The art provides numerous assays particularly useful for
identifying
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previously unknown binding partners for receptor polypeptides of the
invention. For example,
expression cloning using mammalian or bacterial cells, or dihybrid screening
assays can be
used to identify polynucleotides encoding binding partners. As another
example, affinity
chromatography with the appropriate immobilized polypeptide of the invention
can be used to
isolate polypeptides that recognize and bind polypeptides of the invention.
There are a number
of different libraries used for the identification of compounds, and in
particular small
molecule, that modulate (i. e., increase or decrease) biological activity of a
polypeptide of the
invention. Ligands for receptor polypeptides of the invention can also be
identified by adding
exogenous ligands, or cocktails of ligands to two cells populations that are
genetically identical
except for the expression of the receptor of the invention: one cell
population expresses the
receptor of the invention whereas the other does not. The response of the two
cell populations
to the addition of ligands(s) are then compared. Alternatively, an expression
library can be
co-expressed with the polypeptide of the invention in cells and assayed for an
autocrine
response to identify potential ligand(s). As still another example, BIAcore
assays, gel overlay
I S assays, or other methods known in the art can be used to identify binding
partner polypeptides,
including, (1) organic and inorganic chemical libraries, (2) natural product
libraries, and (3)
combinatorial libraries comprised of random peptides, oligonucleotides or
organic molecules.
The role of downstream intracellular signaling molecules in the signaling
cascade of the
polypeptide of the invention can be determined. For example, a chimeric
protein in which the
cytoplasmic domain of the polypeptide of the invention is fused to the
extracellular portion of
a protein, whose ligand has been identified, is produced in a host cell. The
cell is then
incubated with the ligand specific for the extracellular portion of the
chimeric protein, thereby
activating the chimeric receptor. Known downstream proteins involved in
intracellular
signaling can then be assayed for expected modifications i.e. phosphorylation.
Other methods
known to those in the art can also be used to identify signaling molecules
involved in receptor
activity .
5.11.15 ANTI-INFLAMMATORY ACTIVITY
Compositions of the present invention may also exhibit anti-inflammatory
activity. The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in the
inflammatory response, by inhibiting or promoting cell-cell interactions (such
as, for example,
cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the
inflammatory
process, inhibiting or promoting cell extravasation, or by stimulating or
suppressing production
of other factors which more directly inhibit or promote an inflammatory
response.
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Compositions with such activities can be used to treat inflammatory conditions
including
chronic or acute conditions), including without limitation intimation
associated with infection
(such as septic shock, sepsis or systemic inflammatory response syndrome
(SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-
mediated hyperacute
rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory
bowel disease,
Crohn's disease or resulting from over production of cytokines such as TNF or
IL-1.
Compositions of the invention may also be useful to treat anaphylaxis and
hypersensitivity to
an antigenic substance or material. Compositions of this invention may be
utilized to prevent
or treat condition such as, but not limited to, utilized, for example, as part
of methods for the
prevention and/or treatment of disorders involving sepsis, acute pancreatitis,
endotoxin shock,
cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis,
pancreatic cell
damage from diabetes mellitus type 1, graft versus host disease, inflammatory
bowel disease,
inflamation associated with pulmonary disease, other autoimmune disease or
inflammatory
disease, an antiproliferative agent such as for acute or chronic mylegenous
leukemia or in the
prevention of premature labor secondary to intrauterine infections.
5.11.16 LEUKEMIAS
Leukemias and related disorders may be treated or prevented by administration
of a
therapeutic that promotes or inhibits function of the polynucleotides and/or
polypeptides of the
invention. Such leukemias and related disorders include but are not limited to
acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,
promyelocytic,
myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such
disorders, see
Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
5.11.17 NERVOUS SYSTEM DISORDERS
Nervous system disorders, involving cell types which can be tested for
efficacy of
intervention with compounds that modulate the activity of the polynucleotides
and/or
polypeptides of the invention, and which can be treated upon thus observing an
indication of
therapeutic utility, include but are not limited to nervous system injuries,
and diseases or
disorders which result in either a disconnection of axons, a diminution or
degeneration of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient
(including human and non-human mammalian patients) according to the invention
include but
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are not limited to the following lesions of either the central (including
spinal cord, brain) or
peripheral nervous systems:
(i) traumatic lesions, including lesions caused by physical injury or
associated with
surgery, fox example, lesions which sever a portion of the nervous system, or
compression
5 injuries;
(ii) ischemic lesions, in which a Lack of oxygen in a portion of the nervous
system
results in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord
infarction or ischemia;
(iii) infectious lesions, in which a portion of the nervous system is
destroyed or
10 injured as a result of infection, for example, by an abscess or associated
with infection by
human immunodeficiency virus, herpes zoster, or herpes simplex virus or with
Lyme disease,
tuberculosis, syphilis;
(iv) degenerative lesions, in which a portion of the nervous system is
destroyed or
injured as a result of a degenerative process including but not limited to
degeneration
15 associated with Parkinson's disease, Alzheimer's disease, Huntington's
chorea, or amyotrophic
lateral sclerosis;
(v) lesions associated with nutritional diseases or disorders, in which a
portion of
the nervous system is destroyed ox injured by a nutritional disorder or
disorder of metabolism
including but not limited to, vitamin B12 deficiency, folic acid deficiency,
Wernicke disease,
20 tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary
degeneration of the corpus
callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not
limited
to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus,
carcinoma, or
sarcoidosis;
25 (vii) lesions caused by toxic substances including alcohol, lead, or
particular
neurotoxins; and
(viii) demyelinated lesions in which a portion of the nervous system is
destroyed or
injured by a demyelinating disease including but not limited to multiple
sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy or various
etiologies,
30 progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
Therapeutics which are useful according to the invention for treatment of a
nervous
system disorder may be selected by testing for biological activity in
promoting the survival or
differentiation of neurons. For example, and not by way of limitation,
therapeutics which
elicit any of the following effects may be useful according to the invention:
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(i) increased survival time of neurons in culture;
(ii) increased sprouting of neurons in culture or in vivo;
(iii) increased production of a neuron-associated molecule in culture or ih
vivo, e. g. ,
choline acetyltransferase or acetylcholinesterase with respect to motor
neurons; or
(iv) decreased symptoms of neuron dysfunction in vivo.
Such effects may be measured by any method known in the art. In preferred,
non-limiting embodiments, increased survival of neurons may be measured by the
method set
forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting
of neurons may
be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-
82) or Brown et
al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-
associated molecules
may be measured by bioassay, enzymatic assay, antibody binding, Northern blot
assay, etc.,
depending on the molecule to be measured; and motor neuron dysfunction may be
measured by
assessing the physical manifestation of motor neuron disorder, e. g. ,
weakness, motor neuron
conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according
to the
invention include but are not limited to disorders such as infarction,
infection, exposure to
toxin, trauma, surgical damage, degenerative disease or malignancy that may
affect motor
neurons as well as other components of the nervous system, as well as
disorders that
selectively affect neurons such as amyotrophic lateral sclerosis, and
including but not limited to
progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral
sclerosis,
infantile and juvenile muscular atrophy, progressive bulbar paralysis of
childhood
(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and
Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
5.11.1 'OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height, weight,
hair color, eye color, skin, fat to lean ratio or other tissue pigmentation,
or organ or body part
size or shape (such as, for example, breast augmentation or diminution, change
in bone form
or shape); effecting biorhythms or circadian cycles or rhythms; effecting the
fertility of male or
female subjects; effecting the metabolism, catabolism, anabolism, processing,
utilization,
storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins,
minerals,
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co-factors or other nutritional factors or component(s); effecting behavioral
characteristics,
including, without limitation, appetite, libido, stress, cognition (including
cognitive disorders),
depression (including depressive disorders) and violent behaviors; providing
analgesic effects
or other pain reducing effects; promoting differentiation and growth of
embryonic stem cells in
lineages other than hematopoietic lineages; hormonal or endocrine activity; in
the case of
enzymes, correcting deficiencies of the enzyme and treating deficiency-related
diseases;
treatment of hyperproliferative disorders (such as, for example, psoriasis);
immunoglobulin-like activity (such as, for example, the ability to bind
antigens or
complement); and the ability to act as an antigen in a vaccine composition to
raise an immune
response against such protein or another material or entity which is cross-
reactive with such
protein.
5.11.19 IDENTIFICATION OF POLYMORPHISMS
The demonstration of polymorphisms makes possible the identification of such
polymorphisms in human subjects and the pharmacogenetic use of this
information for
diagnosis and treatment. Such polymorphisms may be associated with, e.g.,
differential
predisposition or susceptibility to various disease states (such as disorders
involving
inflammation or immune response) or a differential response to drug
administration, and this
genetic information can be used to tailor preventive or therapeutic treatment
appropriately. For
example, the existence of a polymorphism associated with a predisposition to
inflammation or
autoimmune disease makes possible the diagnosis of this condition in humans by
identifying the
presence of the polymorphism.
Polymorphisms can be identified in a variety of ways known in the art which
all
generally involve obtaining a sample from a patient, analyzing DNA from the
sample,
optionally involving isolation or amplification of the DNA, and identifying
the presence of the
polymorphism in the DNA. For example, PCR may be used to amplify an
appropriate
fragment of genomic DNA which may then be sequenced. Alternatively, the DNA
may be
subjected to allele-specific oligonucleotide hybridization (in which
appropriate oligonucleotides
are hybridized to the DNA under conditions permitting detection of a single
base mismatch) or
to a single nucleotide extension assay (in which an oligonucleotide that
hybridizes immediately
adjacent to the position of the polymorphism is extended with one or more
labeled
nucleotides). In addition, traditional restriction fragment length
polymorphism analysis (using
restriction enzymes that provide differential digestion of the genomic DNA
depending on the
presence or absence of the polymorphism) may be performed. Arrays with
nucleotide
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sequences of the present invention can be used to detect polymorphisms. The
array can
comprise modified nucleotide sequences of the present invention in order to
detect the
nucleotide sequences of the present invention. In the alternative, any one of
the nucleotide
sequences of the present invention can be placed on the array to detect
changes from those
sequences.
Alternatively a polymorphism resulting in a change in the amino acid sequence
could
also be detected by detecting a corresponding change in amino acid sequence of
the protein,
e.g., by an antibody specific to the variant sequence.
5.12 THERAPEUTIC METHODS
The compositions (including polypeptide fragments, analogs, variants and
antibodies or
other binding partners or modulators including antisense polynucleotides) of
the invention have
numerous applications in a variety of therapeutic methods. Examples of
therapeutic
applications include, but are not limited to, those exemplified herein.
5.12.1 EXAMPLE
One embodiment of the invention is the administration of an effective amount
of the
von WillebrandlThrombospondin-like polypeptides or other composition of the
invention to
individuals affected by a disease or disorder that can be modulated by
regulating the Ig
member of the invention. While the mode of administration is not particularly
important,
parenteral administration is preferred. An exemplary mode of administration is
to deliver an
intravenous bolus. The dosage of the von Willebrand/Thrombospondin-like
polypeptides or
other composition of the invention will normally be determined by the
prescribing physician.
It is to be expected that the dosage will vary according to the age, weight,
condition and
response of the individual patient. Typically, the amount of inhibitor
administered per dose
will be in the range of about 0.1 to 25 mg/kg of body weight, with the
preferred dose being
about 0.1 to 10 mg/kg of patient body weight. For parenteral administration,
the von
Willebrand/Thrombospondin-like polypeptides of the invention will be
formulated in an
injectable form combined with a pharmaceutically acceptable parenteral
vehicle. Such vehicles
are well known in the art and examples include water, saline, Ringer's
solution, dextrose
solution, and solutions consisting of small amounts of the human serum
albumin. The vehicle
may contain minor amounts of additives that maintain the isotonicity and
stability of the
polypeptide or other active ingredient. The preparation of such solutions is
within the skill of
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the art. Typically, the cytokine inhibitor will be formulated in such vehicles
at a concentration
of about 1-8 mg/ml to about 10 mg/ml.
5.12.2 ARTHRITIS AND INFLAMMATION
The immunosuppressive effects of the compositions of the invention against
rheumatoid
arthritis is determined in an experimental animal model system. The
experimental model
system is adjuvant induced arthritis in rats, and the protocol is described by
J. Holoshitz, et
at., 1983, Science, 219:56, or by B. Waksman et aL, I963, Int. Arch. Allergy
Appl.
Immunol. , 23:129. Induction of the disease can be caused by a single
injection, generally
intradermally, of a suspension of killed Mycobacterium tuberculosis in
complete Freund's
adjuvant (CFA). The route of injection can vary, but rats may be injected at
the base of the tail
with an adjuvant mixture. The inhibitor is administered in phosphate buffered
solution (PBS) at
a dose of about 1-S mg/kg. The control consists of administering PBS only.
The procedure for testing the effects of the test compound would consist of
1 S intradermally injecting killed Mycobacterium tuberculosis in CFA followed
by immediately
administering the inhibitor and subsequent treatment every other day until day
24. At 14, 15,
18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall
arthritis score may
be obtained as described by J. Holoskitz above. An analysis of the data would
reveal that the
test compound would have a dramatic affect on the swelling of the joints as
measured by a
decrease of the arthritis score.
5.13 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
ADMINISTRATION
A protein or other composition of the present invention (from whatever source
derived,
2S including without limitation from recombinant and non-recombinant sources
and including
antibodies and other binding partners of the polypeptides of the invention)
may be administered
to a patient in need, by itself, or in pharmaceutical compositions where it is
mixed with
suitable carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders. Such a
composition may optionally contain (in addition to protein or other active
ingredient and a
carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known
in the art. The term "pharmaceutically acceptable" means a non-toxic material
that does not
interfere with the effectiveness of the biological activity of the active
ingredient(s). The
characteristics of the carrier will depend on the route of administration. The
pharmaceutical
composition of the invention may also contain cytokines, Iymphokines, or other
hematopoietic
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factors such as M-CSF, GM-CSF, TNF, IL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-
CSF,
thrombopoietin, stem cell factor, and erythropoietin. In further compositions,
proteins of the
invention may be combined with other agents beneficial to the treatment of the
disease or
5 disorder in question. These agents include various growth factors such as
epidermal growth
factor (EGF), platelet-derived growth factor (PDGF), transforming growth
factors (TGF- and
TGF- ), insulin-like growth factor (IGF), as well as cytokines described
herein.
The pharmaceutical composition may further contain other agents which either
enhance
the activity of the protein or other active ingredient or complement its
activity or use in
10 treatment. Such additional factors and/or agents may be included in the
pharmaceutical
composition to produce a synergistic effect with protein or other active
ingredient of the
invention, or to minimize side effects. Conversely, protein or other active
ingredient of the
present invention may be included in formulations of the particular clotting
factor, cytokine,
lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic
factor, or anti-
15 inflammatory agent to minimize side effects of the clotting factor,
cytokine, lymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-
inflammatory agent (such
as IL-lRa, IL-1 Hyl, IL-1 Hy2, anti-TNF, corticosteroids, irrimunosuppressive
agents). A
protein of the present invention may be active in multimers (e.g.,
heterodimers or
homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
20 compositions of the invention may comprise a protein of the invention in
such multimeric or
complexed form.
As an alternative to being included in a pharmaceutical composition of the
invention
including a first protein, a second protein or a therapeutic agent may be
concurrently
administered with the first protein (e.g., at the same time, or at differing
times provided that
25 therapeutic concentrations of the combination of agents is achieved at the
treatment site).
Techniques for formulation and administration of the compounds of the instant
application may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
Easton, PA, latest
edition. A therapeutically effective dose further refers to that amount of the
compound
sufficient to result in amelioration of symptoms, e. g. , treatment, healing,
prevention or
30 amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, a therapeutically effective dose refers to
that ingredient alone.
When applied to a combination, a therapeutically effective dose refers to
combined amounts of
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the active ingredients that result in the therapeutic effect, whether
'administered in combination,
serially or simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically
effective amount of protein or other active ingredient of the present
invention is administered
to a mammal having a condition to be treated. Protein or other active
ingredient of the present
invention may be administered in accordance with the method of the invention
either alone or
in combination with other therapies such as treatments employing cytokines,
lymphokines or
other hematopoietic factors. When co- administered with one or more cytokines,
lymphokines
or other hematopoietic factors, protein or other active ingredient of the
present invention may
be administered either simultaneously with the cytokine(s), lymphokine(s),
other hematopoietic
factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If
administered
sequentially, the attending physician will decide on the appropriate sequence
of administering
protein or other active ingredient of the present invention in combination
with cytokine(s),
lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
5.13.1 ROUTES OF ADMINISTRATION
Suitable routes of administration may, for example, include oral, rectal,
transmucosal,
or intestinal administration; parenteral delivery, including intramuscular,
subcutaneous,
intramedullary injections, as well as intrathecal, direct intraventricular,
intravenous,
intraperitoneal, intranasal, or intraocular injections. Administration of
protein or other active
ingredient of the present invention used in the pharmaceutical composition or
to practice the
method of the present invention can be carried out in a variety of
conventional ways, such as
oral ingestion, inhalation, topical application or cutaneous, subcutaneous,
intraperitoneal,
parenteral or intravenous injection. Intravenous administration to the patient
is preferred.
Alternately, one may administer the compound in a local rather than systemic
manner,
a
for example, via injection of the compound directly into a arthritic joints or
in fibrotic tissue,
often in a depot or sustained release formulation. In order to prevent the
scarring process
frequently occurring as complication of glaucoma surgery, the compounds may be
administered topically, for example, as eye drops. Furthermore, one may
administer the drug
in a targeted drug delivery system, for example, in a liposome coated with a
specific antibody,
targeting, for example, arthritic or fibrotic tissue. The liposomes will be
targeted to and taken
up selectively by the afflicted tissue.
The polypeptides of the invention are administered by any route that delivers
an
effective dosage to the desired site of action. The determination of a
suitable route of
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administration and an effective dosage for a particular indication is within
the level of skill in
the art. Preferably for wound treatment, one administers the therapeutic
compound directly to
the site. Suitable dosage ranges fox the polypeptides of the invention can be
extrapolated from
these dosages or from similar studies in appropriate animal models. Dosages
can then be
adjusted as necessary by the clinician to provide maximal therapeutic benefit.
5.13.2 COMPOSITIONS/FORMULATIONS
Pharmaceutical compositions for use in accordance with the present invention
thus may
be formulated in a conventional manner using one or more physiologically
acceptable carriers
~ comprising excipients and auxiliaries which facilitate processing of the
active compounds into
preparations which can be used pharmaceutically. These pharmaceutical
compositions may be
manufactured in a manner that is itself known, e. g. , by means of
conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or
lyophilizing processes. Proper formulation is dependent upon the route of
administration
chosen. When a therapeutically effective amount of protein or other active
ingredient of the ,
present invention is administered orally, protein or other active ingredient
of the present
invention will be in the form of a tablet, capsule, powder, solution or
elixir. When
administered in tablet form, the pharmaceutical composition of the invention
may additionally
contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule,
and powder contain
from about 5 to 95 % protein or other active ingredient of the present
invention, and preferably
from about 25 to 90 % protein or other active ingredient of the present
invention. When
administered in liquid form, a liquid carrier such as water, petroleum, oils
of animal or plant
origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or
synthetic oils may be
added. The liquid form of the pharmaceutical composition may further contain
physiological
saline solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol,
propylene glycol or polyethylene glycol. When administered in liquid form, the
pharmaceutical composition contains from about 0.5 to 90% by weight of protein
or other
active ingredient of the present invention, and preferably from about 1 to 50
% protein or other
active ingredien of the present invention.
When a therapeutically effective amount of protein or other active ingredient
of the
present invention is administered by intravenous, cutaneous or subcutaneous
injection, protein
or other active ingredient of the present invention will be in the form of a
pyrogen-free,
parenterally acceptable aqueous solution. The preparation of such parenterally
acceptable
protein or other active ingredient solutions, having due regard to pH,
isotonicity, stability, and
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the like, is within the skill in the art. A preferred pharmaceutical
composition for intravenous,
cutaneous, or subcutaneous injection should contain, in addition to protein or
other active
ingredient of the present invention, an isotonic vehicle such as Sodium
Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, Lactated
Ringer's Injection, or other vehicle as known in the art. The pharmaceutical
composition of
the present invention may also contain stabilizers, preservatives, buffers,
antioxidants, or other
additives known to those of skill in the art. For injection, the agents of the
invention may be
formulated in aqueous solutions, preferably in physiologically compatible
buffers such as
Hanks's solution, Ringer's solution, or physiological saline buffer. For
transmucosal
administration, penetrants appropriate to the barrier to be permeated are used
in the
formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining
the
active compounds with pharmaceutically acceptable carriers well known in the
art. Such
carriers enable the compounds of the invention to be formulated as tablets,
pills, dragees, '
capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient
to be treated. Pharmaceutical preparations for oral use can be obtained solid
excipient,
optionally grinding a resulting mixture, and processing the mixture of
granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice starch,
potato starch,
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,
disintegrating agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic
acid or a salt
thereof such as sodium alginate. Dragee cores are provided with suitable
coatings. For this
2S purpose, concentrated sugar solutions may be used, which may optionally
contain gum arabic,
talc, polyvinyl pyrrolidone, 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 identification or to characterize
different
combinations of active compound doses.
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 plasticizer,
such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients in
admixture with filler such
as lactose, binders such as starches, and/or lubricants such as talc or
magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or suspended
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in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In
addition, stabilizers may be added. All formulations for oral administration
should be in
dosages suitable for such administration. For buccal administration, the
compositions may
take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebuliser, with the use of a suitable propellant, e. g.
,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide or
other suitable gas. In the case of a pressurized aerosol the dosage unit may
be determined by
IO providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g. , gelatin for
use in an inhaler or insufflator may be formulated containing a powder mix of
the compound
and a suitable powder base such as lactose or starch. The compounds may be
formulated for
parenteral administration by injection, e.g., by bolus injection or continuous
infusion.
Formulations for injection may be presented in unit dosage form, e, g. , in
ampules or in
multi-dose containers, with an added preservative. The compositions may take
such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of
the active compounds in water-soluble form. 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 oleate or triglycerides, or liposomes. Aqueous injection suspensions may
contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may also contain
suitable stabilizers
or agents which increase the solubility of the compounds to allow for the
preparation of highly
concentrated solutions. Alternatively, the active ingredient may be in powder
form for
constitution with a suitable vehicle, e. g. , sterile pyrogen-free water,
before use.
The compounds rnay also be formulated in rectal compositions such as
suppositories or
retention enemas, e. g. , containing conventional suppository bases such as
cocoa butter or other
glycerides. In addition to the formulations described previously, the
compounds may also be
formulated as a depot preparation. Such long acting formulations may be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection.
Thus, for example, the compounds may be formulated with suitable polymeric or
hydrophobic
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materials (for example as an emulsion in an acceptable oil) or ion exchange
resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a
cosolvent
system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible
organic polymer,
5 and an aqueous phase. The cosolvent system may be the VPD co-solvent system.
VPD is a
solution of 3 % w/v benzyl alcohol, 8 % w/v of the nonpolar surfactant
polysorbate 80, and
65 % wlv polyethylene glycol 300, made up to volume in absolute ethanol. The
VPD
co-solvent system (VPD:SW) consists of VPD diluted 1:1 with a 5 % dextrose in
water
solution. This co-solvent system dissolves hydrophobic compounds well, and
itself produces
10 low toxicity upon systemic administration. Naturally, the proportions of a
co-solvent system
may be varied considerably without destroying its solubility and toxicity
characteristics.
Furthermore, the identity of the co-solvent components may be varied: for
example, other
low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the
fraction size of
polyethylene glycol may be varied; other biocompatible polymers may replace
polyethylene
15 glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may
substitute for
dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds
may be employed. Liposomes and emulsions are well known examples of delivery
vehicles or
carriers for hydrophobic drugs. Certain organic solvents such as
dimethylsulfoxide also may be
employed, although usually at the cost of greater toxicity. Additionally, the
compounds may
20 be delivered using a sustained-release system, such as semipermeable
matrices of solid
hydrophobic polymers containing the therapeutic agent. Various types of
sustained-release
materials have been established and are well known by those skilled in the
art.
Sustained-release capsules may, depending on their chemical nature, release
the compounds for
a few weeks up to over 100 days. Depending on the chemical nature and the
biological
25 stability of the therapeutic reagent, additional strategies for protein or
other active ingredient
stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase
carriers
or excipients. Examples of such carriers or excipients include but are not
limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose derivatives,
gelatin, and
30 polymers such as polyethylene glycols. Many of the active ingredients of
the invention may be
provided as salts with pharmaceutically compatible counterions. Such
pharmaceutically
acceptable base addition salts are those salts which retain the biological
effectiveness and
properties of the free acids and which are obtained by reaction with inorganic
or organic bases
such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine,
dialkylamine,
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monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,
triethanol amine
and the like.
The pharmaceutical composition of the invention may be in the form of a
complex of
the proteins) or other active ingredient of present invention along with
protein or peptide
antigens. The protein and/or peptide antigen will deliver a stimulatory signal
to both B and T
lymphocytes. B lymphocytes will respond to antigen through their surface
immunoglobulin
receptor. T lymphocytes will respond to antigen through the T cell receptor
(TCR) following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins including
those encoded by class I and class II MHC genes on host cells will serve to
present the peptide
antigens) to T lymphocytes. The antigen components could also be supplied as
purified
MHC-peptide complexes alone or with co-stimulatory molecules that can directly
signal T
cells. Alternatively antibodies able to bind surface immunoglobulin and other
molecules on B
cells as well as antibodies able to bind the TCR and other molecules on T
cells can be
combined with the pharmaceutical composition of the invention. The
pharmaceutical
I S composition of the invention may be in the form of a liposome in which
protein of the present
invention is combined, in addition to other pharmaceutically acceptable
carriers, with
amphipathic agents such as lipids which exist in aggregated form as micelles,
insoluble
monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable
lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides,
lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation
of such liposomal
formulations is within the level of skill in the art, as disclosed, for
example, in U.S. Patent
Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are
incorporated herein
by reference.
The amount of protein or other active ingredient of the present invention in
the
pharmaceutical composition of the present invention will depend upon the
nature and severity
of the condition being treated, and on the nature of prior treatments which
the patient has
undergone. Ultimately, the attending physician will decide the amount of
protein or other
active ingredient of the present invention with which to treat each individual
patient. Initially,
the attending physician will administer low doses of protein or other active
ingredient of the
present invention and observe the patient's response. Larger doses of protein
or other active
ingredient of the present invention may be administered until the optimal
therapeutic effect is
obtained for the patient, and at that point the dosage is not increased
further. It is
contemplated that the various pharmaceutical compositions used to practice the
method of the
present invention should contain about 0.01 ~cg to about 100 mg (preferably
about 0.1 ~,g to
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about 10 mg, more preferably about 0.1 ~.g to about 1 mg) of protein or other
active ingredient
of the present invention per kg body weight. For compositions of the present
invention which
are useful for bone, cartilage, tendon or ligament regeneration, the
therapeutic method includes
administering the composition topically, systematically, or locally as an
implant or device.
When administered, the therapeutic composition for use in this invention is,
of course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably be
encapsulated or injected in a viscous form for delivery to the site of bone,
cartilage or tissue
damage. Topical administration may be suitable for wound healing and tissue
repair.
Therapeutically useful agents other than a protein or other active ingredient
of the invention
which may also optionally be included in the composition as described above,
may
alternatively or additionally, be administered simultaneously or sequentially
with the
composition in the methods of the invention. Preferably for bone and/or
cartilage formation,
the composition would include a matrix capable of delivering the protein-
containing or other
active ingredient-containing composition to the site of bone and/or cartilage
damage, providing
a structure for the developing bone and cartilage and optimally capable of
being resorbed into
the body. Such matrices may be formed of materials presently in use for other
implanted
medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential matrices for
the compositions may be biodegradable and chemically defined calcium sulfate,
tricalcium
phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and
polyanhydrides. Other
potential materials are biodegradable and biologically well-defined, such as
bone or dermal
collagen. Further matrices are comprised of pure proteins or extracellular
matrix components.
Other potential matrices are nonbiodegradable and chemically defined, such as
sintered
hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be
comprised of
combinations of any of the above mentioned types of material, such as
polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be
altered in
composition, such as in calcium-aluminate-phosphate and processing to alter
pore size, particle
size, particle shape, and biodegradability. Presently preferred is a 50:50
(mole weight)
copolymer of lactic acid and glycolic acid in the form of porous particles
having diameters
ranging from 150 to 800 microns. In some applications, it will be useful to
utilize a
sequestering agent, such as carboxymethyl cellulose or autologous blood clot,
to prevent the
protein compositions from disassociating from the matrix.
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A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose,
and
carboxymethylcellulose, the most preferred being cationic salts of
carboxymethylcellulose
(CMC). Other preferred sequestering agents include hyaluronic acid, sodium
alginate,
polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and
polyvinyl alcohol).
The amount of sequestering agent useful herein is 0.5-20 wt ~ , preferably 1-
10 wt % based on
total formulation weight, which represents the amount necessary to prevent
desorption of the
protein from the polymer matrix and to provide appropriate handling of the
composition, yet
not so much that the progenitor cells are prevented from infiltrating the
matrix, thereby
providing the protein the opportunity to assist the osteogenic activity of the
progenitor cells. In
further compositions, proteins or other active ingredient of the invention may
be combined
with other agents beneficial to the treatment of the bone and/or cartilage
defect, wound, or
tissue in question. These agents include various growth factors such as
epidermal growth
factor (EGF), platelet derived growth factor (PDGF), transforming growth
factors (TGF- and
TGF- ), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications.
Particularly domestic animals and thoroughbred horses, in addition to humans,
axe desired
patients for such treatment with proteins or other active ingredient of the
present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue
regeneration will be determined by the attending physician considering various
factors which
modify the action of the proteins, e.g., amount of tissue weight desired to be
formed, the site
of damage, the condition of the damaged tissue, the size of a wound, type of
damaged tissue
(e.g., bone), the patient's age, sex, and diet, the severity of any infection,
time of
administration and other clinical factors. The dosage may vary with the type
of matrix used in
the reconstitution arid with inclusion of other proteins in the pharmaceutical
composition. For
example, the addition of other known growth factors, such as IGF I (insulin
like growth factor
I), to the final composition, may also effect the dosage. Progress can be
monitored by periodic
assessment of tissue/bone growth and/or repair, for example, X-rays,
histomorphometric
determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other known
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methods for introduction of nucleic acid into a cell or organism (including,
without limitation,
in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo
in the presence
of proteins of the present invention in order to proliferate or to produce a
desired effect on or
activity in such cells. Treated cells can then be introduced in vivo fox
therapeutic purposes.
5.13.3. EFFECTIVE DOSAGE
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to achieve its
intended purpose. More specifically, a therapeutically effective amount means
an amount
effective to prevent development of or to alleviate the existing symptoms of
the subject being
treated. Determination of the effective amount is well within the capability
of those skilled in
the art, especially in light of the detailed disclosure provided herein. For
any compound used
in the method of the invention, the therapeutically effective dose can be
estimated initially from
appropriate in vitro assays. For example, a dose can be formulated in animal
models to achieve
a circulating concentration range that can be used to more accurately
determine useful doses in
humans. For example, a dose can be formulated in animal models to achieve a
circulating
concentration range that includes the ICso as determined in cell culture (i.
e. , the concentration
of the test compound which achieves a half-maximal inhibition of the protein's
biological
activity). Such information can be used to more accurately determine useful
doses in humans.
A therapeutically effective dose refers to that amount of the compound that
results in
amelioration of symptoms or a prolongation of survival in a patient. Toxicity
and therapeutic
efficacy of such compounds can be determined by standard pharmaceutical
procedures in cell
cultures or experimental animals, e. g. , for determining the LDso (the dose
lethal to 50 % of the
population) and the EDso (the dose therapeutically effective in 50% of the
population). The
dose ratio between toxic and therapeutic effects is the therapeutic index and
it can be expressed
as the ratio between LDso and EDso. Compounds which exhibit high therapeutic
indices are
preferred. The data obtained from these cell culture assays and animal studies
can be used in
formulating a range of dosage for use in human. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the EDso
with little or no
toxicity. The dosage may vary within this range depending upon the dosage form
employed
and the route of administration utilized. The exact formulation, route of
administration and
dosage can be chosen by the individual physician in view of the patient's
condition. See, e. g. ,
Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. l, p.
1. Dosage
amount and interval may be adjusted individually to provide plasma levels of
the active moiety
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which are sufficient to maintain the desired effects, or minimal effective
concentration (MEC).
The MEC will vary for each compound but can be estimated from in vitro data.
Dosages
necessary to achieve the MEC will depend on individual characteristics and
route of
administration. However, HPLC assays or bioassays can be used to determine
plasma
5 concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be
administered using a regimen which maintains plasma levels above the MEC for
10-90 % of the
time, preferably between 30-90% and most preferably between 50-90%. In cases
of local
administration or selective uptake, the effective local concentration of the
drug may not be
10 related to plasma concentration.
An exemplary dosage regimen for polypeptides or other compositions of the
invention
will be in the range of about 0.01 to 100 mg/kg of body weight daily, with the
preferred dose
being about 0.1 to 25 mg/kg of patient body weight daily, varying in adults
and children.
Dosing may be once daily, or equivalent doses may be delivered at longer or
shorter intervals.
15 The amount of composition administered will, of course, be dependent on the
subject
being treated, on the subject's age and weight, the severity of the
affliction, the manner of
administration and the judgment of the prescribing physician.
5.13.4. PACKAGING
20 The compositions may, if desired, be presented in a pack or dispenser
device which
may contain one or more unit dosage forms containing the active ingredient.
The pack may,
for example, comprise metal or plastic foil, such as a blister pack. The pack
or dispenser
device may be accompanied by instructions for administration. Compositions
comprising a
compound of the invention formulated in a compatible pharmaceutical carrier
may also be
25 prepared, placed in an appropriate container, and labeled for treatment of
an indicated
condition.
5.14. ANTIBODIES
Also included in the invention are antibodies to proteins, or fragments of
proteins of the
30 invention. The term "antibody" as used herein refers to immunoglobulin
molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that contain
an antigen binding site that specifically binds (immunoreacts with) an
antigen. Such antibodies
include, but are not limited to, polyclonal, monoclonal, chimeric, single
chain, Fab, Fab~ and
Fcabo2 fragments, and an Fab expression library. In general, an antibody
molecule obtained from
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humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ
from one
another by the nature of the heavy chain present in the molecule. Certain
classes have
subclasses as well, such as IgCn, IgGz., and others. Furthermore, in humans,
the light chain
may be a kappa chain or a lambda chain. Reference herein to antibodies
includes a reference
to all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an
antigen, or a
portion or fragment thereof, and additionally can be used as an immunogen to
generate
antibodies that immunospecifically bind the antigen, using standard techniques
for polyclonal
and monoclonal antibody preparation. The full-length protein can be used or,
alternatively, the
invention provides antigenic peptide fragments of the antigen for use as
immunogens. An
antigenic peptide fragment comprises at least 6 amino acid residues of the
amino acid sequence
of the full length protein, such as an amino acid sequence shown in SEQ ID NO:
4, and
encompasses an epitope thereof such that an antibody raised against the
peptide forms a
a
specific immune complex with the full length protein or with any fragment that
contains the
epitope. Preferably, the antigenic peptide comprises at least 10 amino acid
residues, or at least
15 amino acid residues, or at least 20 amino acid residues, or at least 30
amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of the
protein that are
located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of -related protein that is located on the
surface of the protein,
e.g., a hydrophilic region. A hydrophobicity analysis of the human related
protein sequence
will indicate which regions of a related protein axe particularly hydrophilic
and, therefore, are
likely to encode surface residues useful for targeting antibody production. As
a means for
targeting antibody production, hydropathy plots showing regions of
hydrophilicity and
hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828;
Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is
incorporated herein by
reference in its entirety. Antibodies that are specific for one or more
domains within an
antigenic protein, or derivatives, fragments, analogs or homologs thereof, axe
also provided
herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
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Various procedures known within the art may be used for the production of
polyclonal
or monoclonal antibodies directed against a protein of the invention, or
against derivatives,
fragments, analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory
Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY, incorporated herein by reference). Some of these antibodies are
discussed below.
5.14.1 Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by one or more injections with
the native
protein, a synthetic variant thereof, or a derivative of the foregoing. An
appropriate
immunogenic preparation can contain, for example, the naturally occurring
immunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated
to a second protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation
can further
include an adjuvant. Various adjuvants used to increase the immunological
response include,
but are not limited to, Freund's (complete and incomplete), mineral gels
(e.g., aluminum
hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such
as Bacille
Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory
agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the inununoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for example, by
D. Wilkinson
(The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14,
No. 8 (April 17,
2000), pp. 25-28).
5.14.2 Monoclonal Antibodies
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The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs) of
the monoclonal antibody are identical in all the molecules of the population.
MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a mouse,
hamster, or other appropriate host animal, is typically immunized with an
immunizing agent to
elicit lymphocytes that produce or are capable of producing antibodies that
will specifically
bind to the immunizing agent. Alternatively, the lymphocytes can be immunized
in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof or a fusion
protein thereof. Generally, either peripheral blood lymphocytes are used if
cells of human
origin are desired, or spleen cells or lymph node cells are used if non-human
mammalian
sources are desired. The lymphocytes are then fused with an immortalized cell
line using a
suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-
103).
Immortalized cell lines are usually transformed mammalian cells, particularly
myeloma cells of
rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are
employed.
The hybridoma cells can be cultured in a suitable culture medium that
preferably contains one
or more substances that inhibit the growth or survival of the unfused,
immortalized cells. For
example, if the parental cells Lack the enzyme hypoxanthine guanine
phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances
prevent the
growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell Iines are murine
myeloma
lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San
Diego, California and the American Type Culture Collection, Manassas,
Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the
production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984); Brodeur
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et al., Monoclonal Antibody Production Techniques and Applications, Marcel
Dekker, Inc.,
New York, (1987) pp. 51-63).'
The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in the
art. The binding affinity of the monoclonal antibody can, for example, be
determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
Preferably,
antibodies having a high degree of specificity and a high binding affinity for
the target antigen
are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned
by limiting
dilution procedures and grown by standard methods. Suitable culture media for
this purpose
include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a
mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified from
the culture medium or ascites fluid by conventional immunoglobulin
purification procedures
such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed into
expression vectors,
which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin
protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can
be modified, for example, by substituting the coding sequence for human heavy
and light chain
constant domains in place of the homologous murine sequences (U.S. Patent No.
4,816,567;
Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the
immunoglobulin coding
sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant domains of
an antibody of
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the invention, or can be substituted for the variable domains of one antigen-
combining site of
an antibody of the invention to create a chimeric bivalent antibody.
5.14.2 Humanized Antibodies
5 The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against the
administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')z or
other antigen-
10 binding subsequences of antibodies) that are principally comprised of the
sequence of a human
immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
Humanization can be performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
15 corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In
some instances, Fv framework residues of the human immunoglobulin are replaced
by
corresponding non-human residues. Humanized antibodies can also comprise
residues. which
are found neither in the recipient antibody nor in the imported CDR or
framework sequences.
In general, the humanized antibody will comprise substantially all of at least
one, and typically
20 two, variable domains, in which all or substantially all of the CDR regions
correspond to those
of a non-human immunoglobulin and all or substantially all of the framework
regions are those
of a human immunoglobulin consensus sequence. The humanized antibody optimally
also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta,
Curr. Op.
25 Struct. Biol., 2:593-596 (1992)). '
5.14.3 Human Antibodies
Fully human antibodies relate to antibody molecules in which essentially the
entire
sequences of both the light chain and the heavy chain, including the CDRs,
arise from human
30 genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human
S-cell
hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma
technique to produce human monoclonal antibodies (see Cole, et al. , 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. L1SS, IriC. , pp. 77-96). Human
monoclonal
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86
antibodies may be utilized in the practice of the present invention and may be
produced by
using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or
by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et
al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire. This
approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (BiolTechnolo~y 10, 779-
783 (1992));
Lonberg et al. Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13
(1994)); Fishwild
et al,( Nature Biotechnolo~y 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826
(1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals
which are modified so as to produce fully human antibodies rather than the
animal's
endogenous antibodies in response to challenge by an antigen. (See PCT
publication
W094/02602). The endogenous genes encoding the heavy and tight immunoglobulin
chains in
the nonhuman host have been incapacitated, and active loci encoding human
heavy and light .
chain immunoglobulins are inserted into the host's genome. The human genes are
incorporated, for example, using yeast artificial chromosomes containing the
requisite human
DNA segments. An animal which provides all the desired modifications is then
obtained as
progeny by crossbreeding intermediate transgenic animals containing fewer than
the :full
complement of the modifications. The preferred embodiment of such a nonhuman
animal is a
mouse, and is termed the XenomouseT"'' as disclosed in PCT publications WO
96/33735 and
WO 96/34096. This animal produces B cells which secrete fully human
immunoglobulins.
The antibodies can be obtained directly from the animal after immunization
with an
immunogen of interest, as, for example,. a preparation of a polyclonal
antibody, or
alternatively from immortalized B cells derived from the animal, such as
hybridomas
producing monoclonal antibodies. Additionally, the genes encoding the
immunoglobulins with
human variable regions can be recovered and expressed to obtain the antibodies
directly, or
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can be further modified to obtain analogs of antibodies such as, for example,
single chain Fv
molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S. Patent
No. 5,939,598. It can be obtained by a method including deleting the J segment
genes from at
least one endogenous heavy chain locus in an embryonic stem cell to prevent
rearrangement of
the locus and to prevent formation of a transcript of a rearranged
immunoglobulin heavy chain
locus, the deletion being effected by a targeting vector containing a gene
encoding a selectable
marker; and producing from the embryonic stem cell a transgenic mouse whose
somatic and
germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed
in U.S. Patent No. 5,916,771. It includes introducing an expression vector
that contains a
nucleotide sequence encoding a heavy chain into one mammalian host cell in
culture,
introducing an expression vector containing a nucleotide sequence encoding a
light chain into
another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell
expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
5.14.4 Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Patent
No. 4,946,778). In addition, methods can be adapted for the construction of
Fan expression
libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid
and effective
identification of monoclonal F~e fragments with the desired specificity for a
protein or
derivatives, fragments, analogs or homologs thereof. Antibody fragments that
contain the
idiotypes to a protein antigen may be produced by techniques known in the art
including, but
not limited to: (i) an F(ab~)z fragment produced by pepsin digestion of an
antibody molecule; (ii)
an Fab fragment generated by reducing the disulfide bridges of an F~ab~~z
fragment; (iii) an Fab
fragment generated by the treatment of the antibody molecule with papain and a
reducing agent
and (iv) F~ fragments.
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5.14.5 Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that
have binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for an antigenic protein of the invention. The second
binding target is
any other antigen, and advantageously is a cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas) produce
a potential mixture of ten different antibody molecules, of which only one has
the correct
bispecific structure. The purification of the correct molecule is usually
accomplished by
affinity chromatography steps. Similar procedures are disclosed in WO
93!08829, published
13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part of
the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain
constant region
(CH1) containing the site necessary for light-chain binding present in at
least one of the
fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired,
the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-
transfected into a suitable host organism. For further details of generating
bispecific antibodies
see, for example, Suresh et al., Methods in Enzvmology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair
of antibody molecules can be engineered to maximize the percentage of
heterodimers which are
recovered from recombinant cell culture. The preferred interface comprises at
least a part of
the CH3 region of an antibody constant domain. In this method, one or more
small amino acid
side chains from the interface of the first antibody molecule are replaced
with larger side
chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to
the large side chains) are created on the interface of the second antibody
molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or
threonine). This
provides a mechanism for increasing the yield of the heterodimer over other
unwanted
end-products such as homodimers.
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Bispecific antibodies can be prepared as full length antibodies or antibody
fragments
(e.g. F(ab')z bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a
procedure wherein intact antibodies are proteolytically cleaved to generate
F(ab')z fragments.
These fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide formation. The
Fab' fragments
generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is
mixed with an equimolar amount of the other Fab'-TNB derivative to form the
bispecific
antibody. The bispecific antibodies produced can be used as agents for the
selective
immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')z
molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to directed
chemical coupling in
vitro to form the bispecific antibody. The bispecific antibody thus formed was
able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells, as well as
trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
directly from
recombinant cell culture have also been described. For example, bispecific
antibodies have
been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-
1553 (1992).
The leucine zipper peptides from the Fos and Jun proteins were linked to the
Fab' portions of
two different antibodies by gene fusion. The antibody homodimers were reduced
at the hinge
region to form monomers and then re-oxidized to form the antibody
heterodimers. This
method can also be utilized for the production of antibody homodimers. The
"diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-
6448 (1993)
has provided an alternative mechanism for making bispecific antibody
fragments. The
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable
domain (VL) by a linker which is too short to allow pairing between the two
domains on the
same chain. Accordingly, the VH and VL domains of one fragment are forced to
pair with the
complementary VL and VH domains of another fragment, thereby forming two
antigen-binding
sites. Another strategy for making bispecific antibody fragments by the use of
single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368
(1994).
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Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al. , J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of which
originates in the protein antigen of the invention. Alternatively, an anti-
antigenic arm of an
5 immunoglobulin molecule can be combined with an arm which binds to a
triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or
B7), or Fc
receptors for IgG (Fc yR), such as Fc yRI (CD64), Fc yRII (CD32) and Fc yRIII
(CD 16) so as
to focus cellular defense mechanisms to the cell expressing the particular
antigen. Bispecific
antibodies can also be used to direct cytotoxic agents to cells which express
a particular
10 antigen. These antibodies possess an antigen-binding arm and an arm which
binds a cytotoxic
agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another
bispecific antibody of interest binds the protein antigen described herein and
further binds
tissue factor (TF).
15 5.14.6 Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies
have, for example, been proposed to target immune system cells to unwanted
cells (U.S. Patent
No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373;
EP
20 03089). It is contemplated that the antibodies can be prepared in vitro
using known methods in
synthetic protein chemistry, including those involving crosslinking agents.
For example,
immunotoxins can be constructed using a disulfide exchange reaction or by
forming a thioether
bond. Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
5.14.7 Effector Function Engineering
It can be desirable to modify the antibody of the invention with.respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated can have
improved internalization capability andlor increased complement-mediated cell
killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med., 176: 1191-
1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with
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enhanced anti-tumor activity can also be prepared using heterobifunctional
cross-linkers as
described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have enhanced
complement lysis
and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-
230 (1989).
5.14.8 Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to
a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments thereof), or a
radioactive isotope
(i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been
described above. Enzymatically active toxins and fragments thereof that can be
used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and
PAP-S), rnomordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of
radionuclides are available for the production of radioconjugated antibodies.
Examples include
z~zBi~ ~3iI~ l3IIn, 901, and ~s6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate
HCL), active esters (such as disuccinirnidyl suberate), aldehydes (such as
glutareldehyde), bis-
azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives
(such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene).
For example, a ricin immunotoxin can be prepared as described in Vitetta et
al., Science, 238:
1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of
radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
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using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
5.15 COMPUTER READABLE SEQUENCES
In one application of this embodiment, a nucleotide sequence of the present
invention
can be recorded on computer readable media. As used herein, "computer readable
media"
refers to any medium which can be read and accessed directly by a computer.
Such media
include, but are not limited to: magnetic storage media, such as floppy discs,
hard disc storage
medium, and magnetic tape; optical storage media such as CD-ROM; electrical
storage media
such as RAM and ROM; and hybrids of these categories such as magnetic/optical
storage
media. A skilled artisan can readily appreciate how any of the presently known
computer
readable mediums can be used to create a manufacture comprising computer
readable medium
having recorded thereon a nucleotide sequence of the present invention. As
used herein,
"recorded" refers to a process for storing information on computer readable
medium. A
skilled artisan can readily adopt any of the presently known methods for
recording information
on computer readable, medium to generate manufactures comprising the
nucleotide sequence
information of the present invention.
A variety of data storage structures are available to a skilled artisan for
creating a
computer readable medium having recorded thereon a nucleotide sequence of the
present
invention. The choice of the data storage structure will generally be based on
the means
chosen to access the stored information. In addition, a variety of data
processor programs and
formats can be used to store the nucleotide sequence information of the
present invention on
computer readable medium. The sequence information can be represented in a
word
processing text file, formatted in commercially-available software such as
WordPerfect and
Microsoft Word, or represented in the form of an ASCII file, stored in a
database application,
such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt
any number of
data processor structuring formats (e.g. text file or database) in order to
obtain computer
readable medium having recorded thereon the nucleotide sequence information of
the present
invention.
By providing any of the nucleotide sequences SEQ ID NO: 1 - 3 or a
representative
fragment thereof; or a nucleotide sequence at least 99.9% identical to any of
the nucleotide
sequences of the SEQ ID NO: 1 - 3 in computer readable form, a skilled artisan
can routinely
access the sequence information for a variety of purposes. Computer software
is publicly
available which allows a skilled artisan to access sequence information
provided in a computer
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readable medium. The examples which follow demonstrate how software which
implements
the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et al.,
Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to
identify
open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be
protein
encoding fragments and may be useful in producing commercially important
proteins such as
enzymes used in fermentation reactions and in the production of commercially
useful
metabolites .
As used herein, "a computer-based system" refers to the hardware means,
software
means, and data storage means used to analyze the nucleotide sequence
information of the
present invention. The minimum hardware means of the computer-based systems of
the
present invention comprises a central processing unit (CPU), input means,
output means, and
data storage means. A skilled artisan can readily appreciate that any one of
the currently
available computer-based systems are suitable for use in the present
invention. As stated above,
the computer-based systems of the present invention comprise a data storage
means having
stored therein a nucleotide sequence of the present invention and the
necessary hardware means
and software means for supporting and implementing a search means. As used
herein, "data
storage means" refers to memory which can store nucleotide sequence
information of the
present invention, or a memory access means which can access manufactures
having recorded
thereon the nucleotide sequence information of the present invention.
As used herein, "search means" refers to one or more programs which are
implemented
on the computer-based system to compare a target sequence or target structural
motif with the
sequence information stored within the data storage means. Search means are
used to identify
fragments or regions of a known sequence which match a particular target
sequence or target
motif. A variety of known algorithms are disclosed publicly and a variety of
commercially
available software for conducting search means are and can be used in the
computer-based
systems of the present invention. Examples of such software includes, but is
not limited to,
Smith-Waterman; MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A
skilled artisan can readily recognize that any one of the available algorithms
or implementing
software packages for conducting homology searches can be adapted for use in
the present
computer-based systems. As used herein, a "target sequence" can be any nucleic
acid or
amino acid sequence of six or more nucleotides or two or more amino acids. A
skilled artisan
can readily recognize that the longer a target sequence is, the less likely a
target sequence will
be present as a random occurrence in the database. The most preferred sequence
length of a
target sequence is from about 10 to 100 amino acids or from about 30 to 300
nucleotide
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residues. However, it is well recognized that searches for commercially
important fragments,
such as sequence fragments involved in gene expression and protein processing,
may be of
shorter length.
As used herein, "a target structural motif, " or "target motif, " refers to
any rationally
selected sequence or combination of sequences in which the sequences) are
chosen based on a
three-dimensional configuration which is formed upon the folding of the target
motif. There
are a variety of target motifs known in the art. Protein target motifs
include, but are not
limited to, enzyme active sites and signal sequences. Nucleic acid target
motifs include, but
are not limited to, promoter sequences, hairpin structures and inducible
expression elements
(protein binding sequences).
5.16 EXPRESSION MODULATING SEQUENCES
EMF sequences can be identified within a genome by their proximity to the
ORFs. An
intergenic segment, or a fragment of the intergenic segment, from about 10 to
200 nucleotides
in length, taken 5' from any ORF will modulate the expression of an operably
linked 3' ORF
in a fashion similar to that found with the naturally linked ORF sequence. As
used herein, an
"intergenic segment" refers to the fragments of a genome which are between two
ORF(S)
herein described. Alternatively, EMFs can be identified using known EMFs as a
target
sequence or target motif in the computer-based systems of the present
invention.
The presence and activity of an EMF can be confirmed using an EMF trap vector.
An
EMF trap vector contains a cloning site 5' to a marker sequence. A marker
sequence encodes
an identifiable phenotype, such as antibiotic resistance or a complementing
nutrition
auxotrophic factor, which can be identified or assayed when the EMF trap
vector is placed
within an appropriate host under appropriate conditions. As described above,
an EMF will
modulate the expression of an operably linked marker sequence. A more detailed
discussion of
various marker sequences is provided below. A sequence which is suspected of
being an EMF
is cloned in all three reading frames in one or more restriction sites
upstream from the marker
sequence in the EMF trap vector. The vector is then transformed into an
appropriate host
using known procedures and the phenotype of the transformed host is examined
under
appropriate conditions. As described above, an EMF will modulate the
expression of an
operably linked marker sequence.
5.17 TRIPLE HELIX FORMATION
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In addition, the fragments of the present invention, as broadly described, can
be used to
control gene expression through triple helix formation or antisense DNA or
RNA, both of
which methods are based on the binding of a polynucleotide sequence to DNA or
RNA.
Polynucleotides suitable for use in these methods are usually 20 to 40 bases
in length and are
5 designed to be complementary to a region of the gene involved in
transcription (triple helix -
see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science
15241:456 (1988); and
Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense-
Olmno, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation
optimally results in a
10 shut-off of RNA transcription from DNA, while antisense RNA hybridization
blocks
translation of an mRNA molecule into polypeptide. Both techniques have been
demonstrated
to be effective in model systems. Information contained in the sequences of
the present
invention is necessary for the design of an antisense or triple helix
oligonucleotide.
15 5.18 DIAGNOSTIC ASSAYS AND FITS
The present invention further provides methods to identify the presence or
expression
of one of the ORFs of the present invention, or homolog thereof, in a test
sample,, using a
nucleic acid probe or antibodies of the present invention, optionally
conjugated or otherwise
associated with a suitable label.
20 In general, methods for detecting a polynucleotide of the invention can
comprise
contacting a sample with a compound that binds to and forms a complex with the
polynucleotide for a period sufficient to form the complex, and detecting the
complex, so that
if a complex is detected, a polynucleotide of the invention is detected in the
sample. Such
methods can also comprise contacting a sample under stringent hybridization
conditions with
25 nucleic acid primers that anneal to a polynucleotide of the invention under
such conditions, and
amplifying annealed polynucleotides, so that if a polynucleotide is amplified,
a polynucleotide
of the invention is detected in the sample.
In general, methods for detecting a polypeptide of the invention can comprise
contacting a sample with a compound that binds to and forms a complex with the
polypeptide
30 for a period sufficient to form the complex, and detecting the complex, so
that if a complex is
detected, a polypeptide of the invention is detected in the sample.
In detail, such methods comprise incubating a test sample with one or more of
the
antibodies or one or more of the nucleic acid probes of the present invention
and assaying for
binding of the nucleic acid probes or antibodies to components within the test
sample.
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Conditions for incubating a nucleic acid probe or antibody with a test sample
vary.
Incubation conditions depend on the format employed in the assay, the
detection methods
employed, and the type and nature of the nucleic acid probe or antibody used
in the assay.
One skilled in the art will recognize that any one of the commonly available
hybridization,
amplification or immunological assay formats can readily be adapted to employ
the nucleic
acid probes or antibodies of the present invention. Examples of such assays
can be found in
Chard, T., An Introduction to Radioimmunoassay and Related Techniques,
Elsevier Science
Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al.,
Techniques in
Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983),
Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory
Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam,
The
Netherlands (1985). The test samples of the present invention include cells,
protein or
membrane extracts of cells, or biological fluids such as sputum, blood, serum,
plasma, or
urine. The test sample used in the above-described method will vary based on
the assay
format, nature of the detection method and the tissues, cells or extracts used
as the sample to
be assayed. Methods for preparing protein extracts or membrane extracts of
cells are well
known in the art and can be readily be adapted in order to obtain a sample
which is compatible
with the system utilized.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
Specifically, the invention
provides a compartment kit to receive, in close confinement, one or more
containers which
comprises: (a) a first container comprising one of the probes or antibodies of
the present
invention; and (b) one or more other containers comprising one or more of the
following: wash
reagents, reagents capable of detecting presence of a bound probe or antibody.
In detail, a compartment kit includes any kit in which reagents are contained
in separate
containers. Such containers include small glass containers, plastic containers
or strips of
plastic or paper. Such containers allows one to efficiently transfer reagents
from one
compartment to another compartment such that the samples and reagents are not
cross-contaminated, and the agents or solutions of each container can be added
in a quantitative
fashion from one compartment to another. Such containers will include a
container which will
accept the test sample, a container which contains the antibodies used in the
assay, containers
which contain wash reagents (such as phosphate buffered saline, Tris-buffers,
etc.), and
containers which contain the reagents used to detect the bound antibody or
probe. Types of
detection reagents include labeled nucleic acid probes, labeled secondary
antibodies, or in the
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alternative, if the primary antibody is labeled, the enzymatic, or antibody
binding reagents
which are capable of reacting with the labeled antibody. One skilled in the
art will readily
recognize that the disclosed probes and antibodies of the present invention
can be readily
incorporated into one of the established kit formats which are well known in
the art.
5.19 MEDICAL IMAGING
The novel polypeptides and binding partners of the invention are useful in
medical
imaging of sites expressing the molecules of the invention (e.g., where the
polypeptide of the
invention is involved in the immune response, for imaging sites of
inflammation or infection).
See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778. Such methods involve
chemical attachment
of a labeling or imaging agent, administration of the labeled polypeptide to a
subject in a
pharmaceutically acceptable carrier, and imaging the labeled polypeptide in
vivo at the target
site.
5.20 SCREENING ASSAYS
Using the isolated proteins and polynucleotides of the invention, the present
invention
further provides methods of obtaining and identifying agents which bind to a
polypeptide
encoded by an ORF corresponding to any of the nucleotide sequences set forth
in the SEQ ID
NO: 1 - 3, or bind to a specific domain of the polypeptide encoded by the
nucleic acid. In
detail, said method comprises the steps of:
(a) contacting an agent with an isolated protein encoded by an ORF of the
present
invention, or nucleic acid of the invention; and
(b) determining whether the agent binds to said protein or said nucleic acid.
In general, therefore, such methods for identifying compounds that bind to a
polynucleotide of the invention can comprise contacting a compound with a
polynucleotide of
the invention for a time sufficient to form a polynucleotide/compound complex,
and detecting
the complex, so that if a polynucleotide/compound complex is detected, a
compound that binds
to a polynucleotide of the invention is identified.
Likewise, in general, therefore, such methods for identifying compounds that
bind to a
polypeptide of the invention can comprise contacting a compound with a
polypeptide of the
invention for a time sufficient to form a polypeptide/compound complex, and
detecting the
complex, so that if a polypeptide/compound complex is detected, a compound
that binds to a
polynucleotide of the invention is identified.
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Methods for identifying compounds that bind to a polypeptide of the invention
can also
comprise contacting a compound with a polypeptide of the invention in a cell
for a time
sufficient to form a polypeptide/compound complex, wherein the complex drives
expression of
a receptor gene sequence in the cell, and detecting the complex by detecting
reporter gene
S sequence expression, so that if a polypeptidelcompound complex is detected,
a compound that
binds a polypeptide of the invention is identified.
Compounds identified via such methods can include compounds which modulate the
activity of a polypeptide of the invention (that is, increase or decrease its
activity, relative to
activity observed in the absence of the compound). Alternatively, compounds
identified via
such methods can include compounds which modulate the expression of a
polynucleotide of the
invention (that is, increase or decrease expression relative to expression
levels observed in the
absence of the compound). Compounds, such as compounds identified via the
methods of the
invention, can be tested using standard assays well known to those of skill in
the art for their
ability to modulate activity/expression.
The agents screened in the above assay can be, but are not limited to,
peptides,
carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents
can be selected
and screened at random or rationally selected or designed using protein
modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical
agents
and the like are selected at random and are assayed for their ability to bind
to the protein
encoded by the ORF of ,the present invention. Alternatively, agents may be
rationally selected
or designed. As used herein, an agent is said to be "rationally selected or
designed" when the
agent is chosen based on the configuration of the particular protein. For
example, one skilled
in the art can readily adapt currently available procedures to generate
peptides, pharmaceutical
agents and the like capable of binding to a specific peptide sequence in order
to generate
rationally designed antipeptide peptides, for example see Hurby et al.,
Application of Synthetic
Peptides: Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H.
Freeman, NY
(1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or
pharmaceutical
agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as
broadly
described, can be used to control gene expression through binding to one of
the ORFs or
EMFs of the present invention. As described above, such agents can be randomly
screened or
rationally designed/selected. Targeting the O1RF or EMF allows a skilled
artisan to design
sequence specific or element specific agents, modulating the expression of
either a single ORF
or multiple ORFs which rely on the same EMF for expression control. One class
of DNA
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binding agents are agents which contain base residues which hybridize or form
a triple helix
formation by binding to DNA or RNA. Such agents can be based on the classic
phosphodiester, ribonucleic acid backbone, or can be a variety of sulfliydryl
or polymeric
derivatives which have base attachment capacity.
Agents suitable for use in these methods usually contain 20 to 40 bases and
are
designed to be complementary to a region of the gene involved in transcription
(triple helix -
see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456
(1988); and
Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense-
Okano, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation
optimally results in a
shut-off of RNA transcription from DNA, while antisense RNA hybridization
blocks
translation of an mRNA molecule into polypeptide. Both techniques have been
demonstrated
to be effective in model systems. Information contained in the sequences of
the present
invention is necessary for the design of an antisense or triple helix
oligonucleotide and other
DNA binding agents. Agents which bind to a protein encoded by one of the ORFs
of the
present invention can be used as a diagnostic agent, in the control of
bacterial infection by
modulating the activity of the protein encoded by the ORF. Agents which bind
to a protein
encoded by one of the ORFs of the present invention can be formulated using
known
techniques to generate a pharmaceutical composition.
5.21 USE OF NUCLEIC ACIDS AS PROBES
Another aspect of the subject invention is to provide for polypeptide-specific
nucleic
acid hybridization probes capable of hybridizing with naturally occurring
nucleotide sequences.
The hybridization probes of the subject invention may be derived from any of
the nucleotide
sequences SEQ ID NO: 1 - 3. Because the corresponding gene is only expressed
in a limited
number of tissues, a hybridization probe derived from of any of the nucleotide
sequences SEQ
ID NO: 1 - 3 can be used as an indicator of the presence of RNA of cell type
of such a tissue
in a sample.
Any suitable hybridization technique can be employed, such as, for example, in
situ
hybridization. PCR as described in US Patents Nos. 4,683,195 and 4,965,188
provides
additional uses for oligonucleotides based upon the nucleotide sequences. Such
probes used in
PCR may be of recombinant origin, may be chemically synthesized, or a mixture
of both. The
probe will comprise a discrete nucleotide sequence for the detection of
identical sequences or a
degenerate pool of possible sequences for identification of closely related
genomic sequences.
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Other means for producing specific hybridization probes for nucleic acids
include the
cloning of nucleic acid sequences into vectors for the production of mRNA
probes. Such
vectors are known in the art and are commercially available and may be used to
synthesize
RNA probes in vitro by means of the addition of the appropriate RNA polymerase
as T7 or
SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The
nucleotide
sequences may be used to construct hybridization probes for mapping their
respective genomic
sequences. The nucleotide sequence provided herein may be mapped to a
chromosome or
specific regions of a chromosome using well known genetic and/or chromosomal
mapping
techniques. These techniques include in situ hybridization, linkage analysis
against known
chromosomal markers, hybridization screening with libraries or flow-sorted
chromosomal
preparations specific to known chromosomes, and the like. The technique of
fluorescent in
situ hybridization of chromosome spreads has been described, among other
places, in Verma et
al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New
York
NY. .
Fluorescent in situ hybridization of chromosomal preparations and other
physical
chromosome mapping techniques may be correlated with additional genetic map
data.
Examples of genetic map data can be found in the 1994 Genome Issue of Science
(265:1981fj.
Correlation between the location of a nucleic acid on a physical chromosomal
map and a
specific disease (or predisposition to a specific disease) may help delimit
the region of DNA
associated with that genetic disease. The nucleotide sequences of the subject
invention may be
used to detect differences in gene sequences between normal, carrier or
affected individuals.
The nucleotide sequence may be used to produce purified polypeptides using
well known
methods of recombinant DNA technology. Among the many publications that teach
methods
for the expression of genes after they have been isolated is Goeddel (1990)
Gene Expression
Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego.
Polypeptides
may be expressed in a variety of host cells, either prokaryotic or eukaryotic.
Host cells may
be from the same species from which a particular polypeptide nucleotide
sequence was isolated
or from a different species. Advantages of producing polypeptides by
recombinant DNA
technology include obtaining adequate amounts of the protein for purification
and the
availability of simplified purification procedures.
5.22 PREPARATION OF SEQUENCING CHIPS AND ARRAYS
A basic example is using ~rmers attached to 50 micron surfaces to give a chip
with
dimensions of 3 x 3 mm which can be combined to give an array of 20 x 20 cm.
Another
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example is using 9-mer oligonucleotides attached to 10 x 10 microns surface to
create a 9-mer
chip, with dimensions of 5 x 5 mm. 4000 units of such chips may be used to
create a 30 x 30 cm
array. In an array in which 4,000 to 16,000 oligochips are arranged into a
square array. A plate,
or collection of tubes, as also depicted, may be packaged with the array as
part of the sequencing
kit.
The arrays may be separated physically from each other or by hydrophobic
surfaces. One
possible way to utilize the hydrophobic strip separation is to use technology
such as the Iso-Grid
Microbiology System produced by QA Laboratories, Toronto, Canada.
Hydrophobic grid membrane filters (HGMF) have been in use in analytical food
microbiology for about a decade where they exhibit unique attractions of
extended numerical
range and automated counting of colonies. One commercially available grid is
ISO-GRIDT"" from
QA Laboratories Ltd. (Toronto, Canada) which consists of a square (60 x 60 cm)
ofpolysulfone
polymer (Gelman Tuffryn HT-450, 0.45u pore size) on which is printed a black
hydrophobic ink
grid consisting of 1600 (40 x 40) square cells. HGMF have previously been
inoculated with
bacterial suspensions by vacuum filtration and incubated on the differential
or selective media of
choice.
Because the microbial growth is confined to grid cells of known position and
size on the
membrane, the HGMF functions more like an MPN apparatus than a conventional
plate or
membrane filter. Peterkin et al. (1987) reported that these HGMFs can be used
to propagate and
store genomic libraries when used with a HGMF replicator. One such instrument
replicates
growth from each of the 1600 cells of the ISO-GRID and enables many copies of
the master
HGMF to be made (Peterkin et al., 1987).
Sharpe et al. (1989) also used ISO-GRID HGMF form QA Laboratories and an
automated
HGMF counter (MI-100 Interpreter) and RP-100 Replicator. They reported a
technique for
maintaining and screening many microbial cultures. '
Peterkin and colleagues later described a method for screening DNA probes
using the
hydrophobic grid-membrane filter (Peterkin et al., 1989). These authors
reported methods for
effective colony hybridization directly on HGMFs. Previously, poor results had
been obtained
due to the low DNA binding capacity of the epoxysulfone polymer on which the
HGMFs are
printed. However, Peterkin et al. (1989) reported that the binding of DNA to
the surface of the
membrane was improved by treating the replicated and incubated HGMF
withpolyethyleneimine,
a polycation, prior to contact with DNA. Although this early work uses
cellular DNA
attachment, and has a different objective to the present invention, the
methodology described may
be readily adapted for Format 3 SBH.
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In order to identify useful sequences rapidly, Peterkin et al. (1989) used
radiolabeled
plasmid DNA from various clones and tested its specificity against the DNA on
the prepared
HGMFs. In this way, DNA from recombinant plasmids was rapidly screened by
colony
hybridization against 100 organisms on HGMF replicates which can be easily and
reproducibly
prepared.
Manipulation with small (2-3 mm) chips, and parallel execution of thousands of
the
reactions. The solution of the invention is to keep the chips and the probes
in the corresponding
arrays. In one example, chips containing 250,000 9-mers are synthesized on a
silicon wafer in
the form of 8 x 8 mM plates (15 uM/oligonucleotide, Pease et al., 1994)
arrayed in 8 x 12 format
(96 chips) with a 1 mM groove in between. Probes are added either by
multichannel pipette or
pin array, one probe on one chip. To score all 4000 6-mers, 42 chip arrays
have to be used,
either using different ones, or by reusing one set of chip arrays several
times.
In the above case, using the earlier nomenclature of the application, F=9;
P=6; and F +
P = 15. Chips may have probes of formulaBxNn, where x is a number of specified
bases B; and
n is a number of non specified bases, so that x = 4 to 10 and n = 1 to 4. To
achi:ve more
efficient hybridization, and to avoid potential influence of any support
oligonucleotides, the
specified bases can be surrounded by unspecified bases, thus represented by a
formula such as
(N)nBx(N)m.
5.23 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i. e. , small nucleic acid segments, may be readily prepared
by, for
example, directly synthesizing the oligonucleotide by chemical means, as is
commonly practiced
using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by any of the methods known to
those of
skill in~ the art using any suitable support such as glass, polystyrene or
Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard synthesizers.
Immobilization can be
achieved using passive adsorption (Inouye & Hondo, 1990); using UV light
(Nagata et al., 1985;
Dahlen et al., 1987; Morriey & Collins, 1989) or by covalent binding of base
modified DNA
(Keller et al., 1988; 1989); all references being specifically incorporated
herein.
Another strategy that may be employed is the use of the strong biotin-
streptavidin
interaction as a linker. For example, Broude et al. (1994) describe the use of
Biotinylated probes,
although these are duplex probes, that are immobilized on streptavidin-coated
magnetic beads.
Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this
same linking
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chemistry is applicable to coating any surface with streptavidin. Biotinylated
probes may be
purchased from various sources, such as, e.g., Operon Technologies (Alameda,
CA).
Nunc Laboratories (Naperville, IL) is also selling suitable material that
could be used.
Nunc Laboratories have developed a method by which DNA can be covalently bound
to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface
grafted with
secondary amino groups ( > NH) that serve as bridge-heads for further covalent
coupling.
CovaLink Modules may be purchased fromNunc Laboratories. DNA molecules may be
bound
to CovaLink exclusively at the 5'-end by a phosphoramidate bond, allowing
immobilization of
more than 1 pmol of DNA (Rasmussen et al., 1991).
The use of CovaLink NH strips for covalent binding of DNA molecules at the 5'-
end has
been described (Rasmussen et al., 1991). In this technology, a phosphoramidate
bond is
employed (Chu et al., 1983). This is beneficial as immobilization using only a
single covalent
bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH
secondary
amino groups that are positioned at the end of spacer arms covalently grafted
onto the polystyrene
surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink
NH via an
phosphoramidate bond, the oligonucleotide terminus must have a 5'-end
phosphate group. It is,
perhaps, even possible for biotin to be covalently bound to CovaLink and then
streptavidin used to
bind the probes.
More specifically, the linkage method includes dissolving DNA in water (7.5
ng/ul) and
denaturing for 10 min. at 95° C and cooling on ice for 10 min. Ice-cold
0.1 M
1-methylimidazole, pH 7.0 (1-MeIm~), is then added to a final concentration of
10 mM 1-MeIm~.
A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well)
standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),
dissolved in
10 mM 1-MeIm~, is made fresh and 25 u1 added per well. The strips are
incubated for 5 hours at
50°C. After incubation the strips are washed using, e.g., Nunc-Immuno
Wash; first the wells are
washed 3 times, then they are soaked with washing solution for 5 min., and
finally they are
washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25 % SDS heated
to 50° C).
It is contemplated that a further suitable method for use with the present
invention is that
described in PCT Patent Application WO 90/03382 (Southern & Maskos),
incorporated herein by
reference. This method of preparing an oligonucleotide bound to a support
involves attaching a
nucleoside 3'-reagent through the phosphate group by a covalentphosphodiester
link to aliphatic
hydroxyl groups carried by the support. The oligonucleotide is then
synthesized on the supported
nucleoside and protecting groups removed from the synthetic oligonucleotide
chain under standard
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conditions that do not cleave the oligonucleotide from the support. Suitable
reagents include
nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
An on-chip strategy for the preparation of DNA probe for the preparation of
DNA probe
arrays may be employed. For example, addressable laser-activated
photodeprotectionmay be
employed in the chemical synthesis of oligonucleotides directly on a glass
surface, as described by
Fodor et al. (/99I), incorporated herein by reference. Probes may also be
immobilized on nylon
supports as described by Van Ness et al. (1991); or linked to Teflon using the
method of Duncan
& Cavalier (1988); all references being specifically incorporated herein.
To link an oligonucleotide to a nylon support, as described by Van Ness et al.
(1991),
requires activation of the nylon surface via alkylation and selective
activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
One particular way to prepare support bound oligonucleotides is to utilize the
light-generated synthesis described by Pease et al., (1994, incorporated
herein by reference).
These authors used current photolithographic techniques to generate arrays of
immobilized
oligonucleotide probes (DNA chips). These methods, in which light is used to
direct the synthesis
of oligonucleotide probes in high-density, miniaturized arrays, utilize
photolabile 5'-protected
N acyl-deoxynucleoside phosphoramidites, surface linker chemistry and
versatile combinatorial
synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes
may be generated in
this manner and then used in the advantageous Format 3 sequencing, as
described in WO
95/09248, incorporated herein by reference.
5.24 PREPARATION OF NUCLEIC ACID FRAGMENTS
The nucleic acids to be sequenced may be obtained from any appropriate source,
such as
cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid
or YAC
inserts, and RNA, including mRNA without any amplification steps. For example,
Sambrooket
al. (1989) describes three protocols for the isolation of high molecular
weight DNA from
mammalian cells (p. 9.14-9.23).
DNA fragments may be prepared as clones in M13, plasmid or lambda vectors
and/or
prepared directly from genomic DNA or cDNA by PCR or other amplification
methods. Samples
may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA
samples may be
prepared in 2-500 ml of final volume.
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The nucleic acids would then be fragmented by any of the methods known to
those of skill
in the art including, for example, using restriction enzymes as described at
9.24-9.28 of
Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.
Low pressure shearing is also appropriate, as described by Schriefer et al.
(1990,
incorporated herein by reference). In this method, DNA samples are passed
through a small
French pressure cell at a variety of low to intermediate pressures. A lever
device allows
controlled application of low to intermediate pressures to the cell. The
results of these studies
indicate that low-pressure shearing is a useful alternative to sonic and
enzymatic DNA
fragmentation methods.
One particularly suitable way for fragmenting DNA is contemplated to be that
using the
two base recognition endonuclease, CviJI, described by Fitzgerald et al. (
1992). These authors
described an approach for the rapid fragmentation and fractionation of DNA
into particular sizes
that they contemplated to be suitable for shotgun cloning and sequencing. The
present inventor
envisions that this will also be particularly useful for generating random,
but relatively small,
fragments of DNA for use in the present sequencing technology.
The restriction endonuclease CviJT normally cleaves the recognition sequence
PuGCPy
between the G and C to leave blunt ends. Atypical reaction conditions, Which
alter the specificity
of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments
form the small
molecule pUCl9 (2688 base pairs). Fitzgerald et al. (1992) quantitatively
evaluated the
randomness of this fragmentation strategy, using a CviJI** digest of pUCl9
that was size
fractionated by a rapid gel filtration method and directly ligated, without
end repair, to a lac Z
minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI**
restricts pyGCPy
and PuGCPu, in addition to PuGCPy sites, and that new sequence data is
accumulated at a rate
consistent with random fragmentation.
As reported in the literature, advantages of this approach compared to
sonication and
agarose gel fractionation include: smaller amounts of DNA are required (0.2
0.5 ug instead of
2-5 ug); and fewer steps are involved (no preligation, end repair, chemical
extraction, or agarose
gel electrophoresis and elution are needed). These advantages are also
proposed to be of use
when preparing DNA for sequencing by Format 3.
Irrespective of the manner in which the nucleic acid fragments are obtained or
prepared, it
is important to denature the DNA to give single stranded pieces available for
hybridization. This
is achieved by incubating the DNA solution for 2 5 minutes at 80-90°C.
The solution is then
cooled quickly to 2° C to prevent renaturation of the DNA fragments
before they are contacted
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with the chip. Phosphate groups must also be removed from genomic DNA by
methods known in
the art.
5.25 PREPARATION OF DNA ARRAYS
Arrays may be prepared by spotting DNA samples on a support such as a nylon
membrane. Spotting may be performed by using arrays of metal pins (the
positions of which
correspond to an array of wells in a microtiter plate) to repeated by transfer
of about 20 n1 of a
DNA solution to a nylon membrane. By offset printing, a density of dots higher
than the density
of the wells is achieved. One to 25 dots may be accommodated in 1 mrr~,
depending on the type
of label used. By avoiding spotting in some preselected number of rows and
columns, separate
subsets (subarrays) may be formed. Samples in one subarray may be the same
genomic segment
of DNA (or the same gene) from different individuals, or may be different,
overlapped genomic
clones. Each of the subarrays may represent replica spotting of the same
samples. In one
example, a selected gene segment may be amplified from 64 patients. For each
patient, the
amplified gene segment may be in one 96-well plate (all 96 wells containing
the same sample). A
plate for each of the 64 patients is prepared. By using a 96-pin device, all
samples may be spotted
on one 8 x 12 cm membrane. Subarrays may contain 64 samples, one from each
patient. Where
the 96 subarrays are identical, the dot span may be 1 mrri and there may be a
1 mm space
between subarrays.
Another approach is to use membranes or plates (available from NUNC,
Naperville,
Illinois) which may be partitioned by physical spacers e.g. a plastic grid
molded over the
membrane, the grid being similar to the sort of membrane applied to the bottom
ofmultiwell
plates, or hydrophobic strips. A fixed physical spacer is not preferred for
imaging by exposure to
flat phosphor-storage screens or x-ray films.
The present invention is illustrated in the following examples. Upon
consideration of the
present disclosure, one of skill in the art will appreciate that many other
embodiments and
variations may be made in the scope of the present invention. Accordingly, it
is intended that the
broader aspects of the present invention not be limited to the disclosure of
the following
examples. The present invention is not to be limited in scope by the
exemplified embodiments
which are intended as illustrations of single aspects of the invention, and
compositions and
methods which are functionally equivalent are within the scope of the
invention. Indeed,
numerous modifications and variations in the practice of the invention are
expected to occur to
those skilled in the art upon consideration of the present preferred
embodiments. Consequently,
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the only limitations which should be placed upon the scope of the invention
are those which
appear in the appended claims.
All references cited within the body of the instant specification are hereby
incorporated by
reference in their entirety.
6.0 EXAMPLES
EXAMPLE 1
Isolation of SEO ID NO:1 from a cDNA Librar of Human Fetal Brain
A plurality of novel nucleic acids were obtained from a cDNA library prepared
from
human fetal brain (Hyseq clone identification number 15258924) using standard
PCR,
sequencing by hybridization sequence signature analysis, and Sanger sequencing
techniques.
The inserts of the library were amplified with PCR using primers specific for
vector sequences
flanking the inserts. These samples were spotted onto nylon membranes and
interrogated with
oligonucleotide probes to give sequence signatures. The clones were clustered
into groups of
similar or identical sequences, and single representative clones were selected
from each group
for gel sequencing. The 5' sequence of the amplified inserts was then deduced
using the
reverse M13 sequencing primer in a typical Sanger sequencing protocol. PCR
products were
purified and subjected to fluorescent dye terminator cycle sequencing. Single-
pass gel
sequencing was done using a 377 Applied Biosystems (ABI) sequences. The insert
was
identified as a novel sequence not previously obtained from this library and
not previously
reported in public databases. This sequence is designated as SEQ ID NO: 1 in
the attached
sequence listing.
EXAMPLE 2
ASSEMBLAGE OF SEO ID NO: 2
The nucleic acid of the present invention, designated as SEQ ID NO: 2 was
assembled
using an EST sequence as a seed. Then a recursive algorithm was used to extend
the seed EST
into an extended assemblage, by pulling additional sequences from different
databases (i.e.,
Hyseq's database containing EST sequences, dbEST version 114, gb pri 114, and
UniGene
version 101) that belong to this assemblage. The algorithm terminated when
there was no
additional sequences from the above databases that would extend the
assemblage. Inclusion of
component sequences into the assemblage was based on a BLASTN hit to the
extending
assemblage with BLAST score greater than 300 and percent identity greater than
95 % .
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The nearest neighbor result for the assembled contig was obtained by a FASTA
version 3
search against Genpept release 114, using FASTXY algorithm. FASTXY is an
improved version
of FASTA alignment which allows in-codon frame shifts. The nearest neighbor
result showed the
closest homologue for each assemblage from Genpept (and contains the
translated amino acid
S sequences for which the assemblage encodes). The nearest neighbor results is
set forth below:
Accession No. Description Smith- % Identity
Waterman
Score
298047 Homo sapiens dJ162H14.1316 36.757
(FIBULIN 1 like protein)
A polypeptide was predicted to be encoded by SEQ ID NO: 2 as set forth below.
The
polypeptide was predicted using a software program called FASTY (available
from
http://fasta.bioch.virginia.edu) which selects a polypeptide based on a
comparison of translated
novel polynucleotideto knownpolypeptides~(W.R. Pearson, Methods inEnzymology,
183: 63-98
(1990), herein incorporated by reference).
Predicted PredictedAMINO ACID ENCODED BY SEQ ID NO: 2
beginning end (A=Alanine, C=Cysteine, D=Aspartic Acid,
E=
nucleotidenucleotideGlutamic Acid, F=Phenylalanine, G=Glycine,
location location H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine,
correspond-correspond-M=Methionine, N=Asparagine, P=Proline,
ing to ing to Q=Glutamine, R=Arginine, S=Serine, T=Threonine,
first last
amino acidamino V=Valine, W=Tryptophan, Y=Tyrosine,
acid
residue residue X=Unknown, *=Stop Codon, /=possible
of of nucleotide
amino acidamino deletion, \=possible nucleotide insertion)
acid
segment segment
,
2 542 FVGHCVNTEGGFVCERGPGMRVSADRHSCQDTD
ECLGTPCQQRCKNSIGSYKCSCRTGFHLHGNRHSC
V/DYTPRIPLCSPIFLAAFAPLDVNECRRPLERRVC
HHSCHNTGGSFLCTCRPGFRLRADRVSCE/DFPES
RAGPICHPATPVTPVQEICYCCLLRPHGLPCAQDID
LLLGLQGHQ
EXAMPLE 3
ASSEMBLAGE OF SEQ ID NO: 3
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Assembly of novel nucleotide sequence of SEQ ID NO: 3 was accomplished by
using
an EST sequence SEQ ID NO: 1 as a seed. The seed was extended by using
software
programs such as BLAST and Hyseq proprietary software to pull additional
sequences from
Hyseq's proprietary database containing EST sequences and by gel sequencing
(377 Applied
S Biosystems (ABI) sequencer) using primers to extend both S' and 3' ends.
Inclusion of
component sequences into the assemblage was based on BLAST scores greater than
1000 and a
p-value of p-3 (depending on the length of homology).
EXAMPLE 4
A. Expression of SEQ ID NO: 4-7 in cells
Chinese Hamster Ovary (CHO) cells or other suitable cell types are grown in
DMEM
(ATCC) and 10 % fetal bovine serum (FBS) (Gibco) to 70 % confluence. Prior to
transfection
the media is changed to DMEM and O.S % FCS. Cells are transfected with cDNAs
for SEQ
ID NO: 3 with pBGal vector by the FuGENE-6 transfection reagent (Boehringer).
In
1S summary, 4 ~,1 of FuGENE-6 is diluted in 100 ~,l of DMEM and incubated for
S minutes.
Then, this is added to 1 pg of DNA and incubated for 1S minutes before adding
it to a 3S mm
dish of CHO cells. The CHO cells are incubated at 37° C with S% CO2.
After 24 hours,
media and cell lysates are collected, centrifuged and dialyzed against assay
buffer (1S mM Tris
pH 7.6, 134 mM NaCI, S mM glucose, 3 mM CaClz and MgClz.
B. Expression Study Using SEO ID NO: 3
The expression of SEQ ID NO: 3 in various tissues is analyzed using a
semi-quantitative polymerase chain reaction-based technique. Human cDNA
libraries are used
as sources of expressed genes from tissues of interest (adult bladder, adult
brain, adult heart,
adult kidney, adult lymph node, adult liver, adult lung, adult ovary, adult
placenta, adult
2S rectum, adult spleen, adult testis, bone marrow, thymus, thyroid gland,
fetal kidney, fetal
liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocyte and
macrophage). Gene-specific
primers are used to amplify portions of the SEQ ID NO: 3 sequence from the
samples.
Amplified products are separated on an agarose gel, transferred and chemically
linked to a
nylon filter. The filter is then hybridized with a radioactively labeled (33P-
dCTP)
double-stranded probe generated from SEQ ID NO: 3 using a Klenow polymerase,
random-prime method. The filters are washed (high stringency) and used to
expose a
phosphorimaging screen for several hours. Bands indicate the presence of cDNA
including
SEQ ID NO: 3 sequences in a specific library, and thus mRNA expression in the
corresponding cell type or tissue.
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110
7.0 EQUIVALENTS
Although particular embodiments have been disclosed herein in detail, this has
been
done by way of example for purposes of illustration only, and is not intended
to be limiting
with respect to the scope of the appended claims which follow. In particular,
it is
contemplated by the inventors that various substitutions, alterations, and
modifications may be
made to the invention without departing from the spirit and scope of the
invention as defined
by the claims. The choice of nucleic acid starting material, clone of
interest, or library type is
believed to be a matter of routine for a person of ordinary skill in the art
with knowledge of
the embodiments described herein. Other aspects, advantages, and modifications
considered to
be within the scope of the following claims.
110
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SEQUENCE LISTING
<110> HYSEQ, Inc.
Boyle, Bryan J.
Mize, Nancy K.
Arterburn, Matthew C.
Tang, Y. Tom
Liu, Chenghua
Drmanac, Radoje T.
<120> METHODS AND MATERIALS RELATING TO NOVEL VON
WILLEBRAND/THROMBOSPONDIN FACTOR-LIKE PROTEIN
<130> HYS-15
<140> not yet assigned
<141> 2000-12-22
<150> 09/602,543
<151> 2000-06-21
<150> 09/552,317
<151> 2000-04-25
<l50> 09/488,725
<151> 2000-Ol-21
<160> 7
<170> PatentIn Ver. 2.1
<210> 1
<211> 416
<212> DNA
<213> Homo Sapiens
<400> 1
tttcgtgggc cactgtgtga acacagaagg tgggtttgtg tgctagtgtg ggccgggcat 60
gcagctgtct gccgaccgcc acagctgcca agacactgac gaatgcctag ggactccctg 120
tcagcagaga tgtaaaaaca gcattggcag ctacaagtgt tcctgtcgaa ctggcttcca 280
ccttcatggc aaccggcact cctgtgtaga tacaccccaa ggattcctct ctgctcaccc 240
attttcttgg ctgcctttgc tccccttgat gtaaacgagt gtcggaggcc attggagagg 300
cgagtctgtc accattcctg ccacaacacc gtgggcagct tcctatgcac atgccgacct 360
ggcttcaggc tccgagctga ccgcgtgtcc tgtgaagctt tcccgaaagc cgtgct 416
<210> 2
<211> 541
<212> DNA
<213> Homo Sapiens
<400> 2
tttcgtgggc cactgtgtga acacagaagg tgggtttgtg tgcgagcgtg ggccgggcat 60
gcgggtgtct gccgaccgcc acagctgcca agacactgac gaatgcctag ggactccctg 120
tcagcagaga tgtaaaaaca gcattggcag ctacaagtgt tcctgtcgaa ctggcttcca 180
ccttcatggc aaccggcact cctgtgtaga tacaccccaa ggattcctct ctgctcaccc 240
attttcttgg ctgcctttgc tccccttgat gtaaacgagt gtcggaggcc attggagagg 300
cgagtctgtc accattcctg ccacaacacc gggggcagct ttctatgcac atgccgacct 360
ggcttcaggc tccgagctga ccgcgtgtcc tgtgaagctt tcccgaaagc cgagctggcc 420
ccatttgcca tcctgcaacc ccggtgacac ccgtacaaga ggctactgtt gcttgctgag 480
gccgcacggc ctgccctgtg cccaggacat agacctcctt ctggggctcc agggacatca 540
g 541
1
CA 02395770 2002-06-21
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<210> 3
<211> 2075
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (454)..(1161)
<400> 3
gggccactgt gtgaacacag aaggtgggtt tgtgtgcgag tgtgggccgg gcatgcagct 60
gtctgccgac cgccacagct gccaagacac tgacgaatgc ctagggactc cctgtcagca 120
gagatgtaaa aacagcattg gcagctacaa gtgttcctgt cgaactggct tccaccttca 180
tggcaaccgg cactcctgtg tagatacacc ccaaggattc ctctctgctc acccattttc 240
ttggctgcct ttgctcccct tgatgtaaac gagtgtcgga ggccattgga gaggcgagtc 300
tgtcaccatt cctgccacaa caccgtgggc agcttcctat gcacatgccg acctggcttc 360
aggctccgag ctgaccgcgt gtcctgtgaa gctttcccga aagccgtgct ggccccatct 420
gccatcctgc aaccccggca acacccgtcc aag atg ctt ctg ttg ctt cct gag 474
Met Leu Leu Leu Leu Pro Glu
1 5
gcc ggc cgg cct gcc ctg tcc cca gga cat agc cct cct tct ggg get 522
Ala Gly Arg Pro Ala Leu Ser Pro Gly His Ser Pro Pro Ser Gly Ala
Z5 20
cca ggg ccc cca gcc gga gtc agg acc acc cgc ctg cca tct ccc acc 570
Pro Gly Pro Pro Ala Gly Val Arg Thr Thr Arg Leu Pro Ser Pro Thr
25 30 35
CCa Cga Cta CCC aCa tCC tCC CCt tCt gCC CCt gtg tgg ctg ctg tCC 618
Pro Arg Leu Pro Thr Ser Ser Pro Ser Ala Pro Val Trp Leu Leu Ser
40 45 50 55
acc ctg ctg gcc acc cca gtg cct act gcc tcc ctg ctg ggg aac ctc 666
Thr Leu Leu Ala Thr Pro Val Pro Thr Ala Ser Leu Leu Gly Asn Leu
60 65 70
aga ccc ccc tca ctc ctt cag ggg gag gtg atg ggg acc cct tcc tca 714
Arg Pro Pro Ser Leu Leu Gln Gly Glu Val Met Gly Thr Pro Ser Ser
75 80 85
ccc agg ggc cct gag tcc ccc cga ctg gca gca ggg ccc tct ccc tgc 762
Pro Arg Gly Pro Glu Ser Pro Arg Leu Ala Ala Gly Pro Ser Pro Cys
90 95 100
tgg cac ctg gga gcc atg cat gaa tca agg agt cgc tgg aca gag cct 810
Trp His Leu Gly Ala Met His Glu Ser Arg Ser Arg Trp Thr Glu Pro
105 110 115
ggg tgt tcc cag tgc tgg tgc gag gac ggg aag gtg acc tgt gaa aag 858
Gly Cys Ser Gln Cys Trp Cys Glu Asp Gly Lys Val Thr Cys Glu Lys
120 125 130 135
gtg agg tgt gaa get get tgt tcc cac cca att ccc tcc aga gat ggt 906
Val Arg Cys Glu Ala Ala Cys Ser His Pro Ile Pro Ser Arg Asp Gly
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140 145 150
ggg tgc tgc cca tcg tgc aca ggc tgt ttt cac agt ggt gtc gtc cga 954
Gly Cys Cys Pro Ser Cys Thr Gly Cys Phe His Ser Gly Val Val Arg
155 160 165
get gaa ggg gat gtg ttt tca cct ccc aat gag aac tgc acc gtc tgt 1002
Ala Glu Gly Asp Val Phe Ser Pro Pro Asn Glu Asn Cys Thr Val Cys
170 175 180
gtc tgt ctg get gga aac gtg tcc tgc atc tct cct gag tgt cct tct 1050
Val Cys Leu Ala Gly Asn Val Ser Cys Ile Ser Pro Glu Cys Pro Ser
185 190 195
ggc ccc tgt cag acc ccc ccc aca gac gga ttg ctg tac ttg tgt tcc 1098
Gly Pro Cys Gln Thr Pro Pro Thr Asp Gly Leu Leu Tyr Leu Cys Ser
200 205 210 215
agt gag atg cta ttt cca cgg ccg gtg gta cgc aga cgg ggc tgt gtt 1146
Ser Glu Met Leu Phe Pro Arg Pro Val Val Arg Arg Arg Gly Cys Val
220 225 230
cag tgg ggg tgg tga cgagtgtacc acctgtgttt gccagaatgg ggaggtggag 1201
Gln Trp Gly Trp
235
tgctccttca tgccctgccc tgagctggcc tgcccccgag aagagtggcg gctggcccct 1261
gggcagtgtt gcttcacctg ccaggagccc acaccctcga caggctgctc tcttgacgac 1321
aacggggttg agtttccgat tggacagatc tggccgcctg gggacccctg ggagttatgc 1381
atctgccagg gcggggaggc aggggccggt gtcaccggtg gggtcttggt aaagttcctc 1441
cttcctccag gcagatggct cggtgagctg caagagcaca gactgtgtgg actcctgccc 1501
tcacccaatc cgtatccctg gtcagtgctg cccagactgt tcagcaggct gcacctacac 1561
aggcagaatc ttctataaca acgagacctt cccgtctgtg ctggacccat gtctgagctg 1621
CatCtgCCtg ctgggctcag cggcctgttc ccccgtggac tgccccatca cctgtaccta 1681
ccctttccac ctgacgggga gtgctgcccc gtgtgccgag actgcaacta cgagggaagg 1741
aaggtggcga atggccaggt gttcaccttg gatgatgaac cctgcacccg gtgcacgtgc 1801
cagctgggag aggtgagctg tgagaaggtt ccctgccagc gggcctgtgc cgaccctgcc 1861
ctgcttcctg gggactgctg ctcttcctgt ccagattccc tgtctcctct ggaagaaaag 1921
caggggctct cccctcacgg aaatgtggca ttcagcaaag ctggtcggag cctgcatgga 1981
gacactgagg cccctgtcaa ctgtagctcc tgtCCtgggC CCCCgaCagC atCaCCCtCg 2041
aggccggtgc ttcaactcct ccagctgtat tcgc 2075
<210> 4
<211> 235
<212> PRT
<213> Homo Sapiens
<400> 4
3
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Met Leu Leu Leu Leu Pro Glu Ala Gly Arg Pro Ala Leu Ser Pro Gly
1 5 10 15
His Ser Pro Pro Ser Gly Ala Pro Gly Pro Pro Ala Gly Val Arg Thr
20 25 30
Thr Arg Leu Pro Ser Pro Thr Pro Arg Leu Pro Thr Ser Ser Pro Ser
35 40 45
Ala Pro Val Trp Leu Leu Ser Thr Leu Leu Ala Thr Pro Val Pro Thr
50 55 60
Ala Ser Leu Leu Gly Asn Leu Arg Pro Pro Ser Leu Leu Gln Gly Glu
65 70 75 80
Val Met Gly Thr Pro Ser Ser Pro Arg Gly Pro Glu Ser Pro Arg Leu
85 90 95
Ala Ala Gly Pro Ser Pro Cys Trp His Leu Gly Ala Met His Glu Ser
100 105 110
Arg Ser Arg.Trp Thr Glu Pro Gly Cys Ser Gln Cys Trp Cys Glu Asp
115 120 125
Gly Lys Val Thr Cys Glu Lys Val Arg Cys Glu Ala Ala Cys Ser His
130 135 140
Pro Ile Pro Ser Arg Asp Gly Gly Cys Cys Pro Ser Cys Thr Gly Cys
145 150 155 160
Phe His Ser Gly Val Val Arg Ala Glu Gly Asp Val Phe Ser Pro Pro
165 170 , 175
Asn Glu Asn Cys Thr Val Cys Val Cys Leu Ala Gly Asn Val Ser Cys
180 185 190
Ile Ser Pro Glu Cys Pro Ser Gly Pro Cys Gln Thr Pro Pro Thr Asp
195 200 205
Gly Leu Leu Tyr Leu Cys Ser Ser Glu Met Leu Phe Pro Arg Pro Val
210 215 220
Val Arg Arg Arg Gly Cys Val Gln Trp Gly Trp
225 230 235
<210> 5
<211> 217
<212> PRT
<213> Homo Sapiens
<400> 5
Pro Pro Ser Gly Ala Pro Gly Pro Pro Ala Gly Val Arg Thr Thr Arg
1 5 10 15
Leu Pro S2r Pro Thr Pro Arg Leu Pro Thr Ser Ser Pro Ser Ala Pro
20 25 30
Val Trp Leu Leu Ser Thr Leu Leu Ala Thr Pro Val Pro Thr Ala Ser
35 40 45
Leu Leu Gly Asn Leu Arg Pro Pro Ser Leu Leu Gln Gly Glu Val Met
50 55 60
Gly Thr Pro Ser Ser Pro Arg Gly Pro Glu Ser Pro Arg Leu Ala Ala
65 70 75 80
Gly Pro Ser Pro Cys Trp His Leu Gly Ala Met His Glu Ser Arg Ser
85 90 95
Arg Trp Thr Glu Pro Gly Cys Ser Gln Cys Trp Cys Glu Asp Gly Lys
100 105 110
Val Thr Cys Glu Lys Val Arg Cys Glu Ala Ala Cys Ser His Pro Ile
115 120 125
4
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Pro Ser Arg Asp Gly Gly Cys Cys Pro Ser Cys Thr Gly Cys Phe His
130 135 140
Ser Gly Val Val Arg Ala Glu Gly Asp Val Phe Ser Pro Pro Asn Glu
145 150 155 160
Asn Cys Thr Val Cys Val Cys Leu Ala Gly Asn Val Ser Cys Ile Ser
165 170 175
Pro Glu Cys Pro Ser Gly Pro Cys Gln Thr Pro Pro Thr Asp Gly Leu
180 185 190
Leu Tyr Leu Cys Ser Ser Glu Met Leu Phe Pro Arg Pro Val Val Arg
195 200 205
Arg Arg Gly Cys Val Gln Trp Gly Trp
210 215
<210> 6
<211> 10
<212> PRT
<213> Homo Sapiens
<400> 6
Cys Trp Cys Glu Asp Gly Lys Val Thr Cys
1 5 ZO
<210> 7
<211> 49
<212> PRT
<213> Homo Sapiens
<400> 7
Ser Pro Cys Trp His Leu Gly<Ala Met His Glu Ser Arg Ser Arg Trp
,,..
1 5 3' 10 15
Thr Glu Pro Gly Cys Ser Gln Cys Trp Cys Glu Asp Gly Lys Val Thr
20 25 30
Cys Glu Lys Val Arg Cys Glu Ala Ala Cys Ser His Pro Ile Pro Ser
35 40 45
Arg
1
1
1
